Human Ste20-like stress activated serine/threonine kinase

ABSTRACT

A novel human signal-transduction kinase polypeptide is described which is expressed at a particularly high level in human leukocytes. A full length cDNA which encodes the novel stress-activated serine/threonine kinase polypeptide is disclosed as well as the interior structural region and the amino acid residue sequence of the native biological molecule. Methods are provided to identify compounds that modulate the biological activity of the human Ste20-like stress-activated serine/threonine signal transduction kinase.

[0001] This is a division of application Ser. No. 09/152,406, filed Sep.14, 1998, pending.

[0002] Priority is claimed under 35 USC § 119(a) from UK Application9719920.2 entitled HUMAN STE20-LIKE STRESS ACTIVATED SERINE/THREONINEKINASE, filed Sep. 19, 1997; the entire disclosure of which isincorporated by reference.

FIELD OF THE INVENTION

[0003] The present invention relates to nucleic acid and amino acidsequences of a novel human Ste20-like stress-activated serine/threoninekinase and to the use of these sequences to identify compounds thatmodulate the signal transduction activity of the native biomolecule. Theinvention is also related to the diagnosis, study, prevention, andtreatment of pathophysiological disorders related to the biologicalmolecule.

BACKGROUND OF THE INVENTION

[0004] Cellular response mechanisms to stress are fundamentallyimportant to the human immune system. Stress responses representcarefully devised cellular defense mechanisms which were developed at anearly point during evolution; evidenced by the fact that biomoleculesimplicated in stress response exhibit remarkable similarity across theanimal kingdom. Welch, W. J., et al., The Stress Response and the ImmuneSystem, Inflammation: Basic Principles and Clinical Correlates, RavenPress, Gallin, J. I., et al., Eds., Second Edition, 41:841 (1992).

[0005] Lymphocyte activation, homing, resistance to target cell lysis,tumor antigenicity, regulation of proto-oncogene transcription, andimmune surveillance are examples of immunologic functions that appear tobe mediated or modulated by stress activated signal transductionmolecules. Siegelman, M., et al., Science, 231:823 (1986); Kusher, D.I., et al., J. Immunol., 145:2925 (1990); Ullrich, S. J., et al., PNAS,83:3121 (1986); Colotta, F., et al., Biochem. Biophys. Res. Commun.,168:1013 (1990); Haire, R. N., et al., J. Cell Biol, 106:883 (1988);Born, W., et al., Immunol. T., 11:40 (1990). The number of preactivatedand MHC class II-restricted autoreactive T-lymphocytes in peripheralblood of patients with rheumatoid arthritis, for example, dramaticallyincreases relative to the levels in healthy individuals. Similarly,peripheral blood T-lymphocytes from patients with inflammatory arthritisproliferate strongly in the absence of exogenous antigen or mitogen.Welch, W. J., et al., The Stress Response and the Immune System,Inflammation: Basic Principles and Clinical Correlates, Raven Press,Gallin, J. I., et al., Eds., Second Edition, Chapter 41, 841 (1992).Moreover, synovitis has been shown to result in the generation ofoxygen-derived free radicals that act to perpetuate tissue damage.Blake, D. R., et al., Hypoxic-Reperfusion Injury in the Inflamed HumanJoint, Lancet, 2:2889 (1989).

[0006] The control of hematopoiesis is a highly regulated process thatresponds to a number of physiological stimuli in the human body.Differentiation, proliferation, growth arrest, or apoptosis of bloodcells depends on the presence of appropriate cytokines and theirreceptors, as well as the corresponding cellular signal transductioncascades. Hu, Mickey C. -T., et al., Genes & Development, 10:2251(1996).Generation of mature leukocytes, for instance, is a highly regulatedprocess which responds to various environmental and physiologicalstimuli. Cytokines cause cell proliferation, differentiation orelimination, each of these processes being dependent on the presence ofappropriate cytokine receptors and the corresponding signal transductionelements. Moreover, the stimulation of quiescent B- and T-lymphocytesoccur via antigen receptors which exhibit remarkable homology tocytokine receptors. Grunicke, Hans H., Signal Transduction Mechanisms inCancer, Springer-Verlag (1995). See also, Suchard, S. J., et al.,Mitogen-Activated Protein Kinase Activation During IgG-DependentPhagocytosis in Human Neutrophils, J. Immunol., 158:4961 (1997).

[0007] Distinct signaling cassettes, each containing a central cascadeof kinases, respond to a variety of positive and negative extracellularstimuli, lead to changes in transcription factor activity andposttranslational protein modifications in mammalian cells. Kiefer, F.,et al., EMBO, Vol. 5, 24:7013 (1996). One such protein kinase cascade,known as the mitogen-activated protein kinase (MAPK) cascade, isactivated as an early event in the response of leukocytes to variousstimuli. Stimulation of this pathway has been observed during growthfactor-induced DNA synthesis, differentiation, secretion, andmetabolism. The MAPK pathway has a critical role in the transduction ofreceptor-generated signals from the membrane to the cytoplasm andnucleus. Graves, J. D., et al., Protein Serine/Threonine Kinases of theMAPK Cascade, Annals New York Academy of Sciences, 766:320 (1995). Ithas been established that sustained activation of the MAPK cascade isnot only required, but it is sufficient to trigger the proliferation ofsome cells and the differentiation of others. Cohen, P., Dissection ofProtein Kinase Cascades That Mediate Cellular Response to Cytokines andCellular Stress, Advances in Pharmacology, Academic Press, Hidaka, H.,et al., Eds., Vol. 36, 15 (1996); Marshall, C. J., Cell, 80:179 (1995).Several interdependent biochemical pathways are activated followingeither stimulation of resting T-lymphocytes through the antigen receptoror stimulation of activated T-lymphocytes through the interleukin-2(IL-2) receptor. Many of the events that occur after the engagement ofeither of these receptors are qualitatively similar, such as theactivation of mitogen-activated protein kinase (MAPK) pathways andpreexisting transcription factors, leading to the expression of specificgrowth-associated genes. Symmetry of the Activation of Cyclin-dependentKinaes in Mitogen and Growth Factor-stimulated T Lymphocytes, Jaime F.Modiano, et al., Annals New York Academy of Sciences, 766:134 (1995).

[0008] Recent evidence suggests that cellular response to stress iscontrolled primarily through events occurring at the plasma membrane,overlapping significantly with those important in initiating mitogenicresponses. Exposure of cells to biological, chemical, or physical stressagents evokes a series of events leading to the activation of a widegroup of genes including transcription factors as well as other geneproducts that are also rapidly and highly induced in response tomitogenic stimulation. The mitogen-activated protein kinase (MAPK)pathway has been shown to be essential for the mitogenic reponse in manysystems. See, e.g., Qin, Y. et al., J.Cancer Res.Clin.Oncol., 120:519(1994). Moreover, due to the fact that most oncogenes encode growthfactors, growth factor receptors, or elements of the intracellularpostreceptor signal-transmission machinery, it is becoming increasinglyapparent that growth factor signal transduction pathways are subject toan elaborate network of positive and negative cross-regulatory inputsfrom other transformation-related pathways. Grunicke, Hans H., SignalTransduction Mechanisms in Cancer, Springer-Verlag (1995). TheHierarchical organization of the MAPK cascade makes integral proteinkinase members particularly good targets for such “cross-talk”. ProteinSerine/Threonine Kinases of the MAPK Cascade, J. D. Graves, et al.,Annals New York Academy of Sciences, 766:320 (1995).

[0009] Initial triggers for inflammation include physical and chemicalagents, bacterial and viral infections, as well as exposure to antigens,superantigens or allergens, all of which have the potential to generateReactive Oxygen Species (ROS) and to thereby activate second messengersignal transduction molecules. Storz, G., et al., TranscriptionalRegulators of Oxidative Stress-Inducible Genes in Prokaryotes andEukaryote, in: Stress-Inducible Cellular Responses, Feige, U., et al.,Eds., Birkhauser Verlag (1996). Reactive oxygen radicals, via damage tomany cellular components including DNA, can cause cell death or, if lesssevere, cell cycle arrest at growth-phase checkpoints. Stress damage notonly activates checkpoint controls but also activates protein kinases,including the stress activated protein kinases (SAPKs), c-Raf-1 andERKs, which are integral components of cytoplasmic signal transduction(MAPK) cascades. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996);Russo, T., et al., J.Biol. Chem., 270:29386 (1995). Considering, interalia, that stress has also been implicated in oxidant injury,atherosclerosis, neurogenerative processes, and aging, elucidation ofthe components of mammalian stress-induced pathways should provide morespecific targets that can be exploited therapeutically. N. J. Holbrook,et al., Stress Inducible Cellular Responses, 273, U. Feige, et al.,Eds., Birkhauser Verlag (1996).

[0010] Evidence has demonstrated that mitogen-activated protein kinase(MAPK) and stress activated protein kinase (SAPK) signal transductionpathways are responsible for triggering biological effects across a widevariety of pathophysiological conditions including conditions manifestedby dysfunctional leukocytes, T-lymphocytes, acute and chronicinflammatory disease, auto-immune disorders, rheumatoid arthritis,osteoarthritis, transplant rejection, macrophage regulation, endothelialcell regulation, angiogenesis, atherosclerosis, fibroblasts regulation,pathological fibrosis, asthma, allergic response, ARDS, atheroma,osteoarthritis, heart failure, cancer, diabetes, obeisity, cachexia,Alzheimer's disease, sepsis, and neurodegeneration. As MAP kinases playa central role in signaling events which mediate cellular response tostress, their inactivation is key to the attenuation of the response. N.J. Holbrook, et al., Stress-Inducible Cellular Responses, 273, Feige,U., et al., Eds., Birkhauser Verlag (1996).

[0011] Despite major efforts to develop new therapeutic approaches AdultRespiratory Distress Syndrome (ARDS) (acute pulmonary inflammationcharacterized by the massive generation of Reactive Oxygen Species (ROS)within the lung), for example, remains lethal for about 50% of affectedpatients. Polla. B. S., et al., Stress Proteins in Inflammation, in:Stress Inducible Cellular Responses, Feige, U., et al. Eds., BirkhauserVerlag (1996). Moreover, the chronic inflammatory disease, rheumatoidarthritis, for instance, is believed to be mediated by actvatedT-lymphocytes that infiltrate the synovial membrane and initiate aseries of inflammatory processes. Panayi, G. S., et al., The Importanceof the T-Cell in Initiating and Maintaining the Chronic Synovitis ofRheumatoid Arthritis, Arthritis Rheum, 35:729 (1992). Accumulatingevidence also indicates that the autoimmune disease multiple sclerosis(MS) is mediated by autoreactive T-lymphocytes. Stinissen, P., et al.,Crit. Rev. Immunol., 17(1): 33 (1997). Autoreactive T-lymphocytes havebeen demonstrated to undergo in vivo activation and clonal expansion inpatients with MS. Zhang, J., et al., J. Mol. Med., 74(11): 653 (1996).In diabetes mellitus, autoreactive T-lymphocytes systematically destroypancreatic islet cells such that they prove incapable of producinginsulin. Another propelling recent development in the implication ofoveractive T-cells is the recognition that a particular subset ofT-lymphocytes appear to be a major culprit in asthma and other allergicdiseases, by responding with undue vigor to apparently harmless invaders(rates of asthma per capita in the developing world have increaseddramatically in the last several decades; doubling in the U.S. since1980). New Clues to Asthma Therapies: Vogel, G., Science, 276:1643(1997).

[0012] Recently, much progress has been made in defining the signaltransduction pathways mediating the cellular response to stress. Pombo,C. M., et al., for instance, report the cloning and characterization ofa human Ste20-like oxidant stress response kinase, SOK-1. The kinase ispositively regulated by phohsphorylation and negatively regulated by itsC-terminal non-catalytic region. Reported data suggests SOK-1 transducessignals in response to oxidative and environmental stress. EMBO, Vol.15, 17:4537 (1996). Other stress-activated protein kinase (SAPK),members of the MAPK family, have been shown to be activated in situ byinflammatory stimuli, including tumor-necrosis factor (TNF) andinterleukin-1. Kyriakis, J. M., etal., Nature, 369:156 (1994); Dérijard,B., etal., Cell, 76:025 (1994); Sánchez, I., et al., Nature, 372:794(1994). See also, Kiefer, F., et al., EMBO, Vol. 5, 24:7013 (1996);Creasy, C. L., et al., J. Biol. Chem., 271: No. 35, 21049 (1996));Creasy, C. L., et al., Gene, 167:303 (1995)); Manser, E., et al.,Nature, 367:40 (1994); Hu, Mickey C. -T., et al., Genes & Development,10:2251(1996); Katz, P., et al., J. Biol. Chem., (1994)); Pombo, C. M.,et al., Nature, 377:750 (1995).

[0013] Integral members of cellular signaling pathways as targets fortherapeutic development, for example, have been the subject to severalreviews. See, e.g., Levitzki, A., Signal-Transduction Therapy: A NovelApproach to Disease Management, Eur. J. Biochem, 226:1 (1994); Powis G.,The Potential for Molecular Oncology to Define New Drug Targets, in: NewMolecular Targets for Cancer Chemotherapy, Workman, P., Kerr D. J.,eds., CRC Press, Boca Raton Fla. (1994). As a result of the efforts ofnumerous laboratories, an impressive list of remarkably specificinhibitors of kinases, for instance, has become available. See, e.g.,Levitzki, A., Tyrphostins: Tyrosine Kinase Blockers as NovelAntiproliferative Agents and Dissectors of Signal Transduction, FASEB;6:3275 (1992); Workman P., et al., Discovery and Design of inhibitors ofOncogenic Tyrosine Kinases, in: New Approaches in Cancer Pharmacology:Drug Design and Development, Springer, Berlin 55 (1994).

[0014] A novel class of pyridinyl imidazoles, CSAIDS [SmithKlineBeecham], for instance, have been developed, that inhibit the productionof the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF-α)in monocytes. The drug has been demonstrated to bind specifically to oneprotein in monocytes, termed CSBP (CSAID-binding protein), which hasbeen isolated, cloned, and sequenced and demonstrated as a MAPK homolog.Lee, J. C., et al., Differential Effects of the Bicyclic Imidazoles onCytokine Synthesis in Human Monocytes and Endothelial Cells, AgentsActions, 41: C191 (1994); A Protein Kinase Involved in the Regulation ofInflammatory Cytokine Biosynthesis, Nature, 372:739 (1994). Moreover, asdemonstrated by the identification of rapamycin as a specific inhibitorof the activation of p70 S6 kinase and the identification of compoundsthat inhibit the EGF receptor protein kinase very potently and thatblock the activation of MAP kinase kinase have demonstrated thatspecific inhibitors of protein kinases can indeed be developed. Alessi,D., et al., A Specific Inhibitor of the Activation of MAP KinaseKinase-1 in vitro and in vivo, J. Biol. Chem., 279:27489 (1995); Fry,D., et al., A Specific Inhibitor of the Epidermal Growth Factor ReceptorTyrosine Kinase, Science, 265:806 (1994); Cohen, P., Dissection ofProtein Kinase Cascades That Mediate Cellular Response to Cytokines andCellular Stress, Intracellular Signal Transduction, Advances inPharmacology, Hidaka, H., et al., Eds., Academic Press, 36:17 (1996).

[0015] Compounds which are able to modulate the activity of specificsignal transduction molecules integral to specific intracellularpathways are expected to have significant potential for the ability tocontrol or attenuate downstream physiological responses. Unfortunately,in spite of the introduction of numerous new drugs during the last threedecades, there is a need for new, more efficient and less toxiccompounds. Accordingly, the ability to identify such compounds is ofparamount importance.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to an isolated and purifiedpolynucleotide molecule, which encodes a polypeptide of a humanstress-activated signal-transduction serine/threonine kinase, or abiologically active derivative thereof comprising a nucleic acidsequence encoding the polypeptide having the sequence substantially asdepicted in SEQ ID NO:3 or a biologically active fragment thereof.Isolated and purified polynucleotides of the present invention includebut are not limited to SEQ ID NO:1 (novel human signal-transductionkinase cDNA) and SEQ ID NO:2 (novel human signal-transduction kinasestructural coding region).

[0017] In addition, the current invention is directed to a purifiedpolypeptide comprising the amino acid sequence substantially as depictedin SEQ ID NO:3 which functions as a human signal-transduction kinasepolypeptide.

[0018] The invention is further directed to an expression vector forexpression of a novel human signal-transduction kinase polypeptide in arecombinant host cell, wherein said vector contains a polynucleotidecomprising a nucleic acid sequence encoding a human signal- transductionkinase polypeptide having the sequence substantially as depicted in SEQID NO:3 or a biologically active derivative thereof.

[0019] Further the invention is directed to a host cell containing anexpression vector for expression of a novel human signal-transductionkinase polypeptide, wherein said vector contains a polynucleotidecomprising a nucleic acid sequence encoding the polypeptide of a humankinase having the sequence substantially as depicted in SEQ ID NO:3 or abiologically active derivative thereof. The invention is also directedto a method for producing a polypeptide having the amino acid sequencesubstantially as depicted in SEQ ID NO:3 by culturing said host cellunder conditions suitable for the expression of said polypeptide, andrecovering said polypeptide from the host cell culture.

[0020] The instant invention is further directed to a method ofidentifying compounds that modulate the activity of a humansignal-transduction kinase, comprising:

[0021] (a) combining a candidate compound modulator of a humansignal-transduction kinase activity with a polypeptide of a humansignal-transduction kinase having the sequence substantially as depictedin SEQ ID NO:3, and

[0022] (b) measuring an effect of the candidate compound modulator onthe kinase activity

[0023] The invention is also directed to method of identifying compoundsthat modulate the activity of a human signal-transduction kinase,comprising:

[0024] (a) combining a candidate compound modulator of a humansignal-transduction kinase activity with a host-cell expressing apolypeptide of a human signal-transduction kinase molecule having asequence substantially as depicted in SEQ ID NO:3, and

[0025] (b) measuring an effect of the candidate compound modulator onthe kinase activity.

[0026] The present invention is also directed to active compoundsidentified by means of the aforementioned methods, wherein saidcompounds modulate the activity of a human signal-transduction kinase.

[0027] Further, the invention is directed to a pharmaceuticalcomposition comprising a compound active in the aforementioned methods,wherein said compound is a modulator of a human signal-transductionkinase.

[0028] Additionally, the invention is directed to a novel treatment of apatient in need of such treatment for a condition which is mediated by ahuman signal-transduction kinase, comprising administration of a humansignal-transduction kinase modulating compound active in theaforementioned method.

[0029] The invention is further directed to an antisense polynucleotidemolecule comprising substantially the complement of SEQ ID NO:2 or abiologically-effective portion thereof, or SEQ ID NO:5 or abiologically-effective portion thereof, as well as a method forinhibiting the expression of a human signal-transduction kinasecomprising administering an effective amount of the antisense molecule.

[0030] The current invention is also drawn toward an antibody specificfor a purified polypeptide comprising the amino acid sequencesubstantially as depicted in SEQ ID NO:3, and a diagnostic compositionfor the identification of a polypeptide sequence comprising the aminoacid sequence substantially as depicted in SEQ ID NO:3.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 shows SEQ ID NO:1 which is a 2322 base cDNA nucleic acidsequence which encodes the novel human stress activated kinasepolypeptide described herein.

[0032]FIG. 2 shows SEQ ID NO:2 which is the 1296 base translatedstructural region, ATG to TGA, of the cDNA nucleic acid sequence whichencodes the novel human stress activated kinase polypeptide describedherein.

[0033]FIG. 3 shows SEQ ID NO:3 which is the 431 amino acid residuesequence of the novel human stress activated kinase polypeptidedescribed herein.

[0034]FIG. 4 shows SEQ ID NO:4 which is the 426 amino acid residuesequence of SOK-1, the recently described human oxidant stress activatedkinase. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996).

[0035]FIG. 5 shows a comparison between the amino acid residue sequenceof the novel human stress activated kinase polypeptide describedherein.(SEQ ID NO:3) (designated CD473313), and the amino acid residuesequences of the SOK-1 stress activated kinase (SEQ ID NO:4). Conservedamino acid residues are boxed. Dashes represent gaps introduced tooptimize the alignment. Sequences shown in this figure were producedusing the multisequence alignment program of DNASTAR software (DNASTARInc, Madison Wis.).

[0036]FIG. 6 shows the result of an autoradiographic assay for thekinase activity of the novel human stress activated kinase polypeptidedescribed herein.

[0037]FIG. 7 shows SEQ ID NO:5 which is a 2322 base antisense nucleicacid sequence which is complementary to SEQ ID NO:1.

[0038]FIG. 8 shows SEQ ID NO:6 and SEQ ID NO:7 which are oligonucleotideprimers used to produce a dominant negative inactive novel human stressactivated kinase (K⁵³→R⁵³) mutant. These primers are used to produce themutant/pT7Blue construct (Statagene, LaJolla, Calif.).

[0039]FIG. 9 shows a Northern blot which identifies a primary transcriptpertaining to the novel human kinase, approximately 2.5 kilobases inlength.

[0040]FIG. 10 shows SEQ ID NO:8 and SEQ ID NO:9 which areoligonucleotide PCR primers used to produce full-length nucleic acidsequence pertaining to the novel human stress activated kinase.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one of skillin the art to which this invention belongs. All publications and patentsreferred to herein are incorporated by reference.

[0042] Nucleic acid sequence as used herein refers to anoligonucleotide, nucleotide or polynucleotide sequence, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be double-stranded or single-stranded whether representing the senseor antisense strand. Similarly, amino acid and/or residue sequence asused herein refers to peptide or protein sequences or portions thereof.

[0043] Purified as used herein refers to molecules, either nucleic acidor amino acid sequences, that are removed from their natural environmentand isolated or separated from at least one other component with whichthey are naturally associated.

[0044] As used herein, a functional derivative of the humansignal-transduction kinase molecule disclosed herein is a compound thatpossesses a biological activity (either functional or structural) thatis substantially similar to SEQ ID NO:3. The term “functionalderivatives” is intended to include the “fragments,” “variants,”“degenerate variants,” “analogs” and “homologues”, and to “chemicalderivatives”. The term “variant” is meant to refer to a moleculesubstantially similar in structure and function to either the entireprotein kinase molecule or to a fragment thereof. A molecule is“substantially similar” to the kinase polypeptide if both molecules havesubstantially similar structures or if both molecules possess similarbiological activity. The term “analog” refers to a moleculesubstantially similar in function to either the entire signaltransduction polypeptide, or to a fragment thereof.

[0045] “Substantially as depicted” as used herein refers to functionalderivative proteins, peptides and DNA sequences that may haveinsignificant changes but perform substantially the same biologicalfunction in substantially the same way.

[0046] Biologically active fragment as used herein includes peptideswhich have been truncated with respect to the N- or C-termini, or both;or the corresponding 5′ or 3′ end, or both, of the correspondingpolynucleotide coding region, which fragments perform substantially thesame biological function or encode peptides which perform substantiallythe same function as the precursor. The term “biologically active” alsorefers to the activity of a homolog or analog entity having structural,regulatory or biochemical functions substantially the same as thenaturally occurring entity.

[0047] Expression vector as used herein refers to nucleic acid vectorconstructions which have components to direct the expression ofheterologous protein coding regions including coding regions of thepresent invention through accurate transcription and translation in hostcells. Expression vectors usually contain a promoter to directpolymerases to transcribe the heterologous coding region, a cloning siteat which to introduce the heterologous coding region, and usuallypolyadenylation signals. Expression vectors include but are not limitedto plasmids, retroviral vectors, viral and synthetic vectors.

[0048] Transformed host cells as used herein refer to cells which havecoding regions of the present invention stably integrated into theirgenome, or episomally present as replicating or nonreplicating entitiesin the form of linear nucleic acid or transcript or circular plasmid orvector.

[0049] The term “modulation” is used herein to refer to the capacity toeither enhance or inhibit a functional property of the human kinase ofthe present invention. Such enhancement or inhibition may be contingenton the occurrence of a specific event, such as activation of a signaltransduction pathway, and/or may be manifest only in particular celltypes.

[0050] A cascade signal transduction mechanism is essentially a conduitfor the transmittal of an external stimulus to the cell nucleus in orderto trigger a distinct pattern of gene expression in response to thestimulation. The extracellular signal is amplified and transduced by aseries of independent sequential covalent modifications, from the plasmamembrane to the nucleus, via distinct phosphorylation steps ofindependently specific cognate cytosolic biomolecules. Proteinphosphorylation is now acknowledged as the most important means of acuteregulation of protein function, signal transduction, and gene expressionin eukaryotic cells. Intracellular biomolecule phosphorylation viaspecific kinases is responsible for switching of cellular activity fromone state to another. It is the major mechanism by which cells respondto extracellular signals such as mitogenic stimulation; biological,chemical, and physical stress, hormones, and growth factors. ProteinPhosphorylation, Hardie, D. G., Oxford Press (1993).

[0051] The protein kinases are a large family of enzymes. Conservedstructural motifs provide clear indications as to how the kinasestransfer the y-phosphate of a purine nucleotide triphosphate to thehydroxyl groups of their protein substrates. There are two mainsubdivisions within the superfamily: the protein-serine/threoninekinases and the protein- tyrosine kinases. The kinase domains thatdefine protein kinases contain 12 conserved subdomains (I-XII) that foldinto a common catalytic core structure, as revealed by the 3-dimensionalstructures of several enzymes. The central core of the catalytic domain,the region with greatest frequency of highly conserved residues,consists of subdomains VI through IX. The most striking indicator ofamino acid specificity is found in subdomain VI, the consensus in thisregion is a strong indicator of serine/threonine specificity. See, e.g.,Hanks, S. K., et al., The Protein Kinase Family: Conserved Features andDeduced Phylogeny of the Catalytic Domains, Science, 241:42 (1988);Hanks, S. K., et al., The Eukaryotic Protein Kinase Superfamily: Kinase(Catalytic) Domain Structure and Classification, FASEB, Ser. Rev., 9:576(1995).

[0052] Protein kinases which have closely related catalytic domains, andthus define a family, represent products of genes that have undergonerelatively recent evolutionary divergence. Clustering appears to be ofpredictive value in the determination of the properties and function ofnovel protein kinases. Accordingly, members of a given family tend alsoto share related functions. This is manifest by similarities in overallstructural topology, mode of regulation, and substrate specificity. See,generally, Hardie, D. G., et al., The Protein Kinase Factsbook, AcademicPress, London (1995).

[0053] Progress has been made by many labs in defining signalingpathways initiated by mitogenic stimuli. Blenis, J., Signal Transductionvia the MAP Kinases, PNAS, 90:5889 (1993). The MAP kinase family ofenzymes have been implicated as common and essential components ofsignaling pathways induced by diverse mitogenic stimuli. Once activated,MAP kinases phosphorylate a number of substrates including transcriptionfactors essential for triggering gene expression required for the growthresponse. Accordingly, the MAP kinases are considered to be potentiallyvaluable pharmacological targets within the growth factor signalingpathways. Hidaka, H., et al., Intracellular Signal Transduction,Advances in Pharmacology, Academic Press (1996).

[0054] Mitogen-activated protein kinases (MAPKs) and their upstreamregulatory kinases comprise functional units that couple upstream inputsignals to a variety of outputs. MAPK cascades have been remarkablyconserved in evolution. The core of these cascades is a three-tieredmodule consisting of an MAPK-extracellular signal-regulated kinasekinase (an MEKK), an MEK and an MAPK or extracellular signal-regulatedkinase (ERK). The defining characteristic of these modules is the MAPKitself. The classical pathway, known as the extracellularsignal-regulated kinase pathway (ERK), is activated by mitogens andgrowth factors. ERK has a regulatory kinase, MAPK kinase or MEK,necessary for activation. This enzyme is in turn regulated by anotherMAPK kinase known as Raf. Analogous with the classical MAPK module aretwo other modules which are activated by cytokines and cellular stressesand which have become known as the stress kinase pathways. The definingMAPKs of these pathways are JNK (SAPK) and P38. JNK is activated by theupstream SEK-1 (MKK4) which is activated by MEKK1 or MLK3 whereas P38 isactivated by MKK3 and MKK6.

[0055] In yeast the MAPK modules operate in a linear manner linkingextracellular signals to functional response, whereas in mammalian cells‘cross-talk’ between modules may be obligatory in some cases (eg. IL-2production by T-lymphocytes). Phosphorylation of transcriptional factors(eg. AP-1, NF-kB, ELK-1, ATF-2) by the terminal MAPKs serve to regulateexpression of key inflammatory genes. Differences in the expression ofthe various kinases between cell types cell types and within in responseto processes in disease will have major impact on how cells respond toextracellular stimuli under physiological and pathological conditions.It is for these reasons that there is very likely to be selectivity forspecific inhibitors of these different kinases for their associatedphysiological role as well as opportunities for therapeuticintervention.

[0056] Phosphorylation of transcriptional factors (eg. AP-1, NF-KB,ELK-1, ATF-2) by the terminal MAPKs serve to regulate expression of keyinflammatory genes. Differences in the expression of the various kinasesbetween cell types in response to processes in disease will have majorimpact on how cells respond to extracellular stimuli under physiologicaland pathological conditions. This should provide opportunities fortherapeutic intervention.

[0057] Studies of the budding and fission yeasts, Saccharomycescerevisiae and Schizosaccharomyces pombe, have been particularlyfruitful in the recognition of protein kinases. Hanks, S. K., et al.,The Eukaryotic Protein Kinase Superfamily, FASEB Ser. Rev., 9:576(1995). Signal transduction pathways connecting cell surface receptorswith each member of the MAPK superfamily in mammalian cells areremarkably similar to those of the budding yeast Saccharomycescerevisiae, in which genetic studies have shown parallel signalingcascades leading to the activation of at least three distinctMAPK-related kinases. Hu, Mickey C. -T., et al., Genes & Development,10:2251(1996); See, e.g., Herskowitcz, I., et al., Cell, 80:187 (1995).

[0058] A component of the pheromone-response pathway in budding yeast,Ste20, represented the first identified member of a new family ofserine/threonine protein kinases. Leberer, E., et al., EMBO, 11:4815(1992); Ramer, S. W., et al., PNAS, 90:452 (1993). Several mammalianhomologs to Ste20 have since been identified, including MST 1 (Creasy,C. L., et al., J. Biol. Chem., 271: No. 35, 21049 (1996)), MST2 (Gene,167:303 (1995)), HPK1 (Kiefer, F., et al., EMBO, Vol. 5, 24:7013(1996)). Recently, mammalian Ste20-like kinases, including p21-activatedprotein kinase (PAK1) (Manser, E., et al., Nature, 367:40 (1994)) andgerminal center kinase (GC kinase) (Katz, P., et al., J. Biol. Chem.,(1994)), have been shown to be capable of activating mammalian MAPKcascades. Pombo, C. M., et al., EMBO, Vol. 15, 17:4537 (1996). See also,U.S. Pat. No. 5,605,825, Human PAK65, issued Feb. 25, 1997. Methodsdescribed in U.S. Pat. No. 5,605,825 are herein incorporated herein byreference.

[0059] The MAP kinases require activation by a MAPK/ERK activatingkinase (MEK). The dual-specificity kinase is capable of phosphorylatingboth tyrosine and serine/threonine residues in proteins. Theproto-oncogene c-Raf-1, for instance, has been shown to encode a proteinacting as a MEK kinase and the pathway Raf→MEK→MAPK is now wellestablished as a major signal transduction pathway for growth factors.Activated MAPK undergoes a translocation to the nucleus where it candirectly phosphorylate and activate a variety of transcription factorsincluding c-Myc, C/EBPβ, p62^(TCF)/Elk-1, ATF02 and c-Jun. Grunicke,Hans H., Signal Transduction Mechanisms in Cancer, Springer-Verlag(1995).

[0060] The MAP kinase (MAPK) cascade is critical in mediating severalintracellular actions. In one pathway a series of protein-proteininteractions is triggered at the plasma membrane that culminate inactivation of the GTP-binding protein Ras. GTP-Ras then interacts withthe protein kinase Raf, recruiting it to the plasma membrane where it isactivated. The activation of Raf is followed by the sequentialactivation of three additional kinases: MAP kinase kinase (MAPKK), MAPK,and MAPK-activated protein (MAPKAP) kinase-1. The activation of MAPK andMAPKAP kinase-1 leads to their translocation from the cytosol to thenucleus where they regulate the activities of transcription factors.Cohen, P., Dissection of Protein Kinase Cascades That Mediate CellularResponse to Cytokines and Cellular Stress, Advances in Pharmacology,Academic Press, Hidaka, H., et al., Eds., Vol. 36, 15 (1996); Marshall,C. J., Cell, 80:179 (1995).

[0061] Recent evidence suggests that cellular response to stress iscontrolled primarily through events occurring at the plasma membrane,overlapping significantly with those important in initiating mitogenicresponses. Exposure of cells to stress agents, as mentioned supra,evokes a series of events leading to the activation of a wide group ofgenes including transcription factors as well as other gene productsthat are also rapidly and highly induced in response to mitogenicstimulation. Pathways which are involved in mediating these cellularresponses rely on the activation of mitogen-activated protein kinases(MAPK) which include extracellular signal-regulated kinases (ERK),stress activated protein kinases (SAPK), c-Jun N-terminal kinases (JNK),and p38/PK/CSBP kinases. These kinases play a key role in the activationof transcription factors and other regulatory proteins involved inactivating gene expression. Phosphorylation enhances complex formationand the serum response element located in the promoters of stressresponse genes such as c-fos. Other regulated proteins includep90^(RSK), activating transcription factor-2 (ATF-2, a cAMP responseelement-binding protein), NF-IL6 (nuclear factor for the activation ofInterleukin 6) and c-Myc. Davis, R. J., The Mitogen-Activated ProteinKinase Signal Transduction Pathway, J. Biol. Chem., 268:1553 (1993); N.J. Holbrook, et al., Stress Inducible Cellular Responses, 273, in:Stress-Inducible Cellular Responses, Feige, U., et al., Eds., BirkhauserVerlag (1996). The mitogen-activated protein kinase (MAPK) pathway hasbeen shown to be essential for the mitogenic reponse in many systems.See, e.g., Qin, Y. et al., J.Cancer Res.Clin.Oncol., 120:519 (1994).Moreover, due to the fact that most oncogenes encode growth factors,growth factor receptors, or elements of the intracellular postreceptorsignal-transmission machinery, it is becoming increasingly apparent thatgrowth factor signal transduction pathways are subject to an elaboratenetwork of positive and negative cross-regulatory inputs from othertransformation-related pathways. Grunicke, Hans H., Signal TransductionMechanisms in Cancer, Springer-Verlag (1995). The Hierarchicalorganization of the MAPK cascade makes integral protein kinase membersparticularly good targets for such “cross-talk”. ProteinSerine/Threonine Kinases of the MAPK Cascade, J. D. Graves, et al.,Annals New York Academy of Sciences, 766:320 (1995).

[0062] Initial triggers for inflammation include physical and chemicalagents, bacterial and viral infections, as well as exposure to antigens,superantigens or allergens, all of which have the potential to generateReactive Oxygen Species (ROS) and to thereby activate second messengersignal transduction molecules. Reactive oxygen species are generatedfrom molecular oxygen and include the free radicals superoxide (—O₂ ⁻),hydroxyl (.OH) and nitric oxide (NO.), as well as non-radicalintermediates such as hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂).During normal cellular respiration, ROS are constantly produced at lowrate in both eukaryotes and prokaryotes. At these low concentrations,ROS can act as second messengers, stimulate cell proliferation, and actas mediators for cell activation. However, during phagocytosis,infection or inflammation, ROS can accumulate to toxic levels whichleads to oxidative stress, and may damage almost all cellularcomponents. All organisms have mechanisms to detoxify the oxidants or torepair the damage caused by ROS, including superoxide dismutases,catalases, peroxidases, glutathione, thioredoxin, and heat shockproteins. The expression of the genes coding these proteins (oxidativestress genes) is induced by changes in the concentrations of ROS,suggesting that cells have developed mechanisms to sense the ROS. Storz,G., et al., Transcriptional Regulators of Oxidative Stress-InducibleGenes in Prokaryotes and Eukaryote, in: Stress-Inducible CellularResponses, Feige, U., et al., Eds., Birkhauser Verlag (1996).

[0063] Isozymes generally:

[0064] The study of isozymes has grown into one of fundamentalsignificance in the investigation of the molecular basis of cellulardifferentiation and morphogenesis. Isozymes represent a metabolic typeof regulation accomplished through the participation of multiple formsof a given enzyme or enzyme subunits that occur within an organism, indifferent cell types, or even within a single cell. In many instancesall forms of the particular enzyme catalyze the same overall reactionbut differ in their dependence on substrate concentration, ioniccofactors, and cellular conditions. Isozymes such as lactatedehydrogenase, for example, occur in five different forms ofapproximately the same molecular weight. Although predominantly inskeletal muscle, lactate dehydrogenase isozymes exist in differentproportions in different tissues such as cardiac and red muscle tissue,as well as embryonic tissues. The relative proportions of the lactatedehydrogenase, for example, set of isozymes in particular tissues isparticularly important in the diagnosis of heart and liver disease. TheA4 and A3B isozymes are elevated in liver disease, therefore the bloodserum factor isozyme levels of lactate dehydrogenase, for instance, areused in clinical diagnoses. Isozymes are now known for a great manydifferent enzymes. Many allosteric enzymes occur as two or more isozymesthat vary in sensitivity to their allosteric modulators. Differentisozymes have accordingly evolved as mechanistic response to biochemicalstress—their presence therefore is a reliable factor in the indicationof precise physiological conditions.

[0065] SOK-1:

[0066] Pombo et al. recently reported the cloning and characterizationof human Ste20/oxidant stress response kinase, SOK-1 which belongs tothe Sps1/GC kinase group of Ste20-like kinases with N-terminal catalyticdomains. The kinase is positively regulated by phohsphorylation andnegatively regulated by its C-terminal non-catalytic region. SOK-1 isactivated relatively specifically by oxidant stress. Reported dataplaces SOK-1 in a stress response pathway and suggest it resembles infunction yeast Ste20s which transduce signals in response toenvironmental stress. EMBO, Vol. 15, 17:4537 (1996). The open readingframe is reported to encode a protein of 426 amino acids and has apredicted M_(r) of 48,041 Da. The kinase domain is located in theN-terminal half of the protein. The reported peptide contains all 11subdomains of serine/threonine kinases. Ste20 related stress-activatedkinases are evidenced to be in proximity to the membrane in thesignaling cascade and therefore are able to provide greater targetopportunity for selective modulation of signal transduction.

[0067] See, Schinkman, K., et al., Cloning and Characterization of aHuman STE20-Like Protein Kinase with Unusual Cofactor Requirements, J.Biol. Chem., 272 (45): 28695-28703 (Nov. 7, 1997); GENBANK locusAF024636 1970 bp mRNA PRI 02-NOV-1997, Definition: Homo sapiensSTE20-like kinase 3 (mst-3) mRNA, NID g2582412.

[0068] Novel Signal-Transduction Kinase:

[0069] A novel human stress-activated signal transductionserine/threonine protein kinase molecule, as well as example nucleicacid sequences which encode therefor, are herein described.

[0070] A cDNA sequence is provided, (SEQ ID NO:1) FIG. 1, whichcomprises the structural coding region of the native human signaltransduction kinase (SEQ ID NO:2) FIG. 2. The 2322bp SEQ ID NO:1contiguous cDNA sequence contains a 1293 bp open reading frame (ORF)with a Kozak initiation sequence at the start methionine. The SEQ IDNO:1 region which encodes the structural biomolecule, (SEQ ID NO:3) FIG.3, extends from the ATG translation initiation codon beginning at baseposition 8 to the CAC final open reading frame codon ending at baseposition 1300 (bases 1301-1303 represent the Opal native terminationcodon TGA). FIG. 2 (SEQ ID NO:2), shows the 1296 base open reading frameincluding the stop codon. The native human homolog of the Ste20-likestress-activated kinase (SEQ ID NO:3) is shown in FIG. 3. The 431 aminoacid residue sequence of the novel protein contains all eleven (11)sudomains found in eukaryotic protein kinases including Ste20-likekinases. The protein has a predicted M_(r) of 47,919 Da, an isolectricpoint of 5.24, and a net charge of −12.68.

[0071] Mitogen-activated protein kinase cascades have been remarkablyconserved in evolution. FIG. 4, for example, shows the 426 amino acidresidue sequence of SOK-1 (SEQ ID NO:4) which is the recently describedhuman MAPK-pathway oxidant (stress) activated kinase. SOK-1 has beencharacterized as a human homolog of the MAPK-pathway yeaststress-activated kinase Ste20. Pombo, C. M., et al., EMBO, Vol. 15,17:4537 (1996). FIG. 5 shows a comparison between the amino acid residuesequence of the human stress activated kinase described herein.(SEQ IDNO:3) (designated CD473313), and the amino acid residue sequences of theSOK-1 stress activated kinase (SEQ ID NO:4). Conserved amino acidresidues are boxed. Dashes represent gaps introduced to optimize thealignment. Sequences shown in this figure were produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.). The novel human stress-activated signal transductionserine/threonine protein kinase molecule (SEQ ID NO:3) has 70% aminoacid residue homology to the previously reported human Ste20-likehomolog SOK-1 (SEQ ID NO:4). Pombo et al. reported SEQ ID NO:4 as ahuman Ste20/oxidant stress response kinase. Ste20 relatedstress-activated kinases, via evidentiary characterization, appear to beclose to the plasma membrane in the signaling cascade and therefore mayhave significant potential to provide greater target opportunity forselective modulation of signal transduction. Pombo, C. M., et al., EMBO,Vol. 15, 17:4537 (1996).

[0072] An example chimeric fusion peptide, comprised of GlutathionineS-transferase (GST) and the novel human kinase described herein, isdemonstrated to phosphorylate myelin basic protein (MBP) as well as toautophosphorylate in vitro—described in EXAMPLE I, as well as theautoradiograph shown in FIG. 6. The catalytic domain of the novelstress-activated kinase (SEQ ID NO:3 residues 24-295) demonstrates 88.6percent homologous identity with the catalytic domain of SOK-1 (SEQ IDNO:4 residues 20-291). The catalytic domain can be further divided into12 subdomains defined by strings of conserved residues. The sequenceGKGSFGEV, for instance, amino acid residue positions 31-38 of SEQ IDNO:3, corresponds to the general consensus GxGxxGxV found in subdomain Iof protein kinases. The subdomain II of protein kinases, identified by aconserved lysine (K) in the tripeptide sequence AxK, is generallyaccepted to be involved in the phosphotransfer reaction. The novelkinase described herein demonstrates this consensus sequence in SEQ IDNO:3 residues 51-53 (AIK). Other functionally important subdomains ofthe protein kinase family are subdomains VI through IX. These particularregions are generally accepted to form the central core of the catalyticsite—characterized by series of highly conserved residues. These domainsinclude the generally conserved residues Asp(D)¹⁶⁶ and Asn(N)¹⁷¹ insubdomain VI and Asp(D)¹⁸⁴, Phe(F)¹⁸⁵, and Gly(G)¹⁸⁶ in subdomain VII;all of which have been implicated in ATP binding (reference amino acidresidue positions are based on positions in the catalytic subunit of aform cAMP-dependent protein kinase). Region VIB contains the consensussequence HRDLxxxN, with D being the heretofore invariant Asp(D)¹⁶⁶. Thenovel human kinase, SEQ ID NO:3 residues 142-149 in this region showHRDIKAAN wherein a conservative substitution of Ile(I) for Leu(L) isapparent. Furthermore, the stress-activated signal transduction kinasemolecule described herein, SEQ ID NO:3, contains the conserved DFGresidues 162-164 of subdomain VII. The Asp(D) is generally accepted tofunctionally orient the γ-phosphate of the ATP for transfer inphosphorylation. Subdomain VIII of the novel kinase contains the highlyconserved APE sequence, with the Glu(E) SEQ ID NO:3 residue 189,corresponding to the invariant Glu(E)²⁰⁸. This particular subdomain isthought to play a critical role in the recognition of substrate binding.Additionally, many kinases are activated by phosphorylation of residuesin subdomain VIII. The consensus sequence DxWS/AxG of subdomain IX isrepresented by amino acid residues 201-206 of SEQ ID NO:3 (DIWSLG). Thisregion is believed to form a large a-helix while the initial Asp(D) ofthe consensus sequence serves to stabilize the catalytic loop byhydrogen bonding.

[0073] Variations:

[0074] The present invention also encompasses variants of the humansignal-transduction kinase molecule SEQ ID NO:3. A preferred variantsubstantially as depicted in SEQ ID NO:3, for instance, is one having atleast 80% amino acid sequence similarity; a more preferred variant isone having at least 90% amino acid sequence similarity; and a mostpreferred variant is one having at least 95% amino acid sequencesimilarity to the kinase molecule amino acid sequence (SEQ ID NO:3) or abiologically active fragment thereof.

[0075] A “variant” of the human kinase molecule of the present inventionmay have an amino acid sequence that is different by one or more amineacid “substitutions”. The variant may have “conservative” changes,wherein a substituted amine acid has similar structural or chemicalproperties, e.g., replacement of leucine with isoleucine. More rarely, avariant may have “nonconservative” changes, e.g., replacement of aglycine with a tryptophan. Similar minor variations may also includeamine acid deletions or insertions, or both. Guidance in determiningwhich and how many amine acid residues may be substituted, inserted ordeleted without abolishing biological or immunological activity may befound using computer programs well known in the art, for example,DNAStar software.

[0076] The present invention relates to nucleic acid (SEQ ID NO:1 andSEQ ID NO:2) and amino acid sequences (SEQ ID NO:3) of the novel humankinase and variations thereof and to the use of these sequences toidentify compounds that modulate the activity of the kinase, asdescribed infra.

[0077] The invention further relates to the use of the humansignal-transduction kinase molecule in expression systems as assays foragonists or antagonists of the biomolecule. The invention also relatesto the diagnosis, study, prevention, and treatment of disease related tothe biological signals transduced by the serine/threonine kinasedescribed herein.

[0078] Polynucleotide sequences which encode the human humansignal-transduction kinase SEQ ID NO:3 or a functionally equivalentderivative thereof may be used in accordance with the present inventionwhich comprise deletions, insertions and/or substitutions of the SEQ IDNO:2 nucleic acid sequence. Biologically active variants of the kinasemolecule of the present invention may also be comprised of deletions,insertions or substitutions of SEQ ID NO:3 amino acid residues. Apurified polynucleotide comprising a nucleic acid sequence encoding thepolypeptide having the sequence substantially as depicted in SEQ ID NO:3or a biologically active fragment thereof is a particularly preferredembodiment of the present invention.

[0079] Amino acid substitutions of SEQ ID NO:3 may be made, forinstance, on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of the kinase is retained.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threoninephenylalanine, and tyrosine.

[0080] Nucleic acid sequences which encode the amino acid sequence ofthe kinase molecule described herein are of an exponential sum due tothe potential substitution of degenerate codons (different codons whichencode the same amino acid). The oligonucleotide sequence selected forheterologous expression is therefore preferably tailored to meet themost common characteristic tRNA codon recognition of the particular hostexpression system used as well known by those skilled in the art.

[0081] Suitable conservative substitutions of amino acids are known tothose of skill in this art and may be made without altering thebiological activity of the resulting polypeptide, regardless of thechosen method of synthesis. The phrase “conservative substitution”includes the use of a chemically derivatized residue in place of anon-derivatized residue provided that such polypeptide displays thedesired binding activity. D-isomers as well as other known derivativesmay also be substituted for the naturally occurring amino acids. See,e.g., U.S. Pat. No. 5,652,369, Amino Acid Derivatives, issued Jul. 29,1997. Substitutions are preferably, although not exclusively, made inaccordance with those set forth in TABLE 1 as follows: TABLE 1 Originalresidue Example conservative substitution Ala (A) Gly; Ser; Val; Leu;Ile; Pro Arg (R) Lys; His; Gln; Asn Asn (N) Gln; His; Lys; Arg Asp (D)Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn;Gln; Arg; Lys Ile (I) Leu; Val; Met; Ala; Phe Leu (L) Ile; Val; Met;Ala; Phe Lys (K) Arg; Gln; His; Asn Met (M) Leu; Tyr; Ile; Phe Phe (F)Met; Leu; Tyr; Val; Ile; Ala Pro (P) Ala, Gly Ser (S) Thr Thr (T) SerTrp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V) Ile; Leu; Met; Phe;Ala

[0082] The nucleotide sequences of the present invention may also beengineered in order to alter a coding sequence for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art,e.g., site-directed mutagenesis to insert new restriction sites, toalter glycosylation patterns, to change codon preference, etc.

[0083] Included within the scope of the present invention are alleles ofthe human signal-transduction kinase molecule of the present invention.As used herein, an “allele” or “allelic sequence” is an alternative formof the kinase molecule described herein. Alleles result from nucleicacid mutations and mRNA splice-variants which produce polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one or many allelic forms. Common mutational changes which giverise to alleles are generally ascribed to natural deletions, additionsor substitutions of amino acids. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

[0084] The present invention relates, in part, to the inclusion of thepolynucleotide encoding the novel human signal-transduction kinasemolecule in an expression vector which can be used to transform hostcells or organisms. Such transgenic hosts are useful for the productionof the protein kinase.

[0085] The nucleic acid sequence also provides for the design ofantisense molecules useful in downregulating, diminishing, oreliminating expression of the genomic nucleotide sequence in cellsincluding leukocytes, endothelial cells, and tumor or cancer cells.

[0086] The human signal-transduction kinase molecule of the presentinvention can also be used in screening assays to identify antagonistsor inhibitors which bind, emulate substrate, or otherwise inactivate orcompete with the biomolecule. The novel kinase can also be used inscreening assays to identify agonists which induce the production of orprolong the lifespan of the molecule in vivo or in vitro.

[0087] The invention also relates to pharmaceutical compounds andcompositions comprising the kinase molecule substantially as depicted inSEQ ID NO:3, or fragments thereof, antisense molecules capable ofdisrupting expression of the naturally occurring gene, and agonists,antibodies, antagonists or inhibitors of the native signal-transductionkinase. These compositions are useful for the prevention or treatment ofconditions associated with abnormal expression of the signaltransduction molecule such as described infra.

[0088] Pharmacological Significance:

[0089] Compounds which are able to modulate the activity of specificsignal transduction molecules integral to specific intracellularpathways are expected to have significant potential for the ability tocontrol or attenuate downstream physiological responses. Significantevidence has been provided that stress kinase activation pathways areresponsible for biological effects across a wide variety of diseaseareas. As MAP kinases play a central role in signaling events whichmediate cellular response to stress; compounds which modulate orinactivate specific integral signal transduction molecules, i.e., thenovel human stress- activated serine/threonine signal transductionkinase molecule described herein, SEQ ID NO:3, and nucleic acidsequences coding therefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, havesignificant potential for the ability attenuate pathophysiologicalresponses. Accordingly, the ability to screen for antagonists andagonists which modulate the activity of the native humanstress-activated serine/threonine signal transduction kinase moleculedescribed herein is significantly valuable toward the identification anddevelopment of therapeutic agents.

[0090] Potential diagnostic and therapeutic applications are readilyapparent for modulators of the human Ste20-like stress-activatedserine/threonine kinase described herein. Areas which are common todisease particularly in need of therapeutic intervention include but arenot limited to pathophysiological disorders manifested by dysfunctionalleukocytes, T-cell activation, acute and chronic inflammatory disease,auto-immune disorders, rheumatoid arthritis, osteoarthritis, transplantrejection, macrophage regulation, endothelial cell regulation,angiogenesis, atherosclerosis, vascular disease, fibroblasts regulation,pathological fibrosis, asthma, allergic response, ARDS, atheroma,osteoarthritis, heart failure, cancer, diabetes, obesity, cachexia,Alzheimer's, sepsis, neurodegeneration, and related disorders.

[0091] Oxidants, mechanical stress, UV irradiation, and immunologicmediators lead to the activation of proinflammatory gene products,apoptosis-related genes, and acute phase response genes. These responsesare the underlying causes of vascular injury and inflammation. In viewof the recent literature, it is generally accepted that MAPK pathwaysincluding the SAPK/JNK and p38 kinase pathways, for example, are centralcomponents in the response of cells to chemical, mechanical andproinflammatory assaults. With such a prominent role in cell activation,stress kinases are likely to play an important role in disease areasincluding, but not limited to, stroke (ischemia reperfusion), ARDS,sepsis, neurodegeneration, and inflammation. During reperfusion ofischemic tissue, for instance, there is a marked increase in stressactivated protein kinase activity leading to c-Jun/ATF-2 activation toenhance gene transcription leading to either apoptosis ordifferentiation and cell repair. In the inflammatory response, TNF-α andIL-1β activate p38, which triggers production of more TNF and IL-1,which amplifies the inflammatory response. This process is thought toplay a role in both septic shock and formation of the atherscleroticplaque. SAPKs are also thought to play a role in the induction ofE-selectin expression in endothelial cells and matrix metalloproteinasesin inflammatory cells, indicating a role for SAPKs in postischemicinjury and tissue remodeling. Moreover, SAPK-p46β₁, a brain-specifickinase, co-localizes with the prominent Alzheimer's disease marker,ALZ-50, suggesting that the proline-directed hyperphosphorylation of tauprotein is catalyzed by this kinase.

[0092] The signal-transduction kinase homolog described herein (SEQ IDNO:3) is believed to transduce cellular response to stressors leading tothe activation of proinflammatory gene products. The deleterious effectsof the mediators of inflammation, including ROS and cytokines, open newavenues for the development of original anti-inflammatory therapies. AsMAP kinases play a central role in signaling events which mediatecellular response to stress, their inactivation or antagonization is keyto the attenuation of the response. Clear evidence has been shown, forinstance, that ERK and JNK pathways are strongly linked to IL-2production. ERK and JNK pathways are clearly established to be requiredfor full T-lymphocyte activation leading to IL-2 gene transcription andT-cell proliferation. Interruption of the signaling process by selectivekinase inhibition is therefore expected to reduce IL-2 production andT-lymphocyte proliferation which would be therapeutically beneficial inchronic inflammatory diseases. MAPKs including stress activated kinasesare expected to play key roles in the pathology of autoimmune diseasesincluding but not limited to rheumatoid arthritis (RA), osteoarthritis(OA), and multiple sclerosis (MS). Accordingly, the novel humanserine/threonine stress-activated kinase signal transduction moleculedescribed herein (SEQ ID NO:3), and nucleic acid sequences codingtherefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significant potentialas a specific targets that can be exploited diagnostically andtherapeutically for the control of dysfunctional leukocytes, includingbut not limited to dysfunctional T-lymphocytes, and in the treatment ofautoimmune disease including rheumatoid arthritis (RA) andosteoarthritis (OA), and in the study, diagnosis, and treatment of acuteand chronic inflammation including but not limited to conditions such asasthma and ARDS as well as other diseases manifested by dysfunctionalleukocytes.

[0093] Excess production of oxidants is responsible for the activationof MAPK pathways. Excess production of oxidants is also common to themajor atherosclerotic risk factors as well as to ischaemic reperfusioninjury. Lipid peroxidation in cell membranes, cytoplasmic free radicals,together with ‘oxidative stress’ lead to activation of AP-1 and NF-kB.The JNK pathway has been established as well as activation of Rasappears to be involved in the responses. Stimulation of macrophages andendothelial cells by LPS or TNF/IL-1 results in the activation of MAPK,JNK and P38 pathways. The selective P38 inhibitor SB203580, for example,has been clearly shown to inhibit production of TNF and IL-1 byLPS-stimulated macrophages as well as TNF, IL-6 and IL-8 byTNF-stimulated HUVECs. Philip Cohen, Dissection of Protein KinaseCascades That Mediate Cellular Response to Cytokines and CellularStress, in: Intracellular Signal Transduction, Advances in Pharmacology,Vol. 36, Academic Press (1996). MAPK pathways are also activated byshear stress. Atherosclerotic lesions develop and progress in areas oflow and unstable shear stress and not in areas exposed to steady shear.Acute changes in shear stress activate MAPK pathways and chronic stressdesensitizes MAPK and NF-kB pathways indicating that these pathways areactivated. Transient shear stress can activate inflammatory genes partlythrough activation of the JNK pathway and AP-1 although the ERK pathwayis also activated. Despite major efforts to develop new therapeuticapproaches Adult Respiratory Distress Syndrome (ARDS) (acute pulmonaryinflammation characterized by the massive generation of Reactive OxygenSpecies (ROS) within the lung) remains lethal for about 50% of affectedpatients. Polla. B. S., et al., Stress Proteins in Inflammation, in:Stress Inducible Cellular Responses, Feige, U., et al. Eds., BirkhauserVerlag (1996). Accordingly, the novel human serine/threonine stress-activated kinase signal transduction molecule described herein (SEQ IDNO:3), and nucleic acid sequences coding therefor, e.g., SEQ ID NO:1 andSEQ ID NO:2, have significant potential as specific targets that can beexploited diagnostically and therapeutically for the control ofpathophysiologies relating to inflammation, dysfunctional macrophages,dysfunctional endothelial cells, and related inflammatory diseasesincluding but not limited to conditions such as atherosclerosis, asthma,allergic response, ARDS, heart failure, Atheroma and related disorders.

[0094] Differentiation, proliferation, growth arrest, or apoptosis ofcells depends on the presence of appropriate cytokines and theirreceptors, as well as the corresponding cellular signal transductioncascades. It remains clear that stress kinase pathways, make criticalcontributions to transformation. In view of the positioning ofraf-MEK1-Erk1/2 downstream of ras, the antiproliferative biologicaleffect of inhibiting signal transduction in the MAP kinase pathway havesignificant therapeutic potential, applicable, for example, but notlimited to tumours harbouring oncogenic ras. Moreover, activation ofstress pathway kinases has been implicated in mediating apoptosis byagents such as TNF in model systems. Evidence regarding the involvementof intracellular signaling pathways in the maintenance of cellularsurvival suggests that survival and mitogenic signals can be separableand that the balance between these signals plays a key role indetermining the fate of transformed cells. There are also indicationsthat perturbation of this balance provides selective apoptosis. It wouldbe critical to achieve optimum selectivity between pathways to achieveapoptosis promotion in tumour cells.

[0095] Unfortunately, in spite of the introduction of numerous new drugsduring the last three decades, only very modest progress can beregistered with regard to both cure or survival rates of cancer patientstreated with anti-tumor agents. Thus, there is a need for new, moreefficient and less toxic compounds. The majority of presently usedanti-tumor agents interfere with the biosynthesis of nucleic acids ortheir intracellular function. Compounds which inhibit uncontrolledgrowth by interfering with mitogenic signal transduction may act ascytostatic rather than cytotoxic drugs. Furthermore, attenuation ofcellular proliferation has frequently been shown to cause tissuedifferentiation. Finally, blockade of mitogenic stimulation in a cellcan result in the inductioin of apoptosis or programmed cell death.Accordingly, the novel human serine/threonine stress-activated kinasesignal transduction molecule described herein (SEQ ID NO:3), and nucleicacid sequences coding therefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, havesignificant potential as a specific targets that can be exploiteddiagnostically and therapeutically to control cancer and theproliferation of tumor cells.

[0096] Activation of members of each of the MAPK pathways has beendemonstrated in response to endothelin, serum, PDGF and TGFβ in varioustypes of ‘fibroblast’. Dominant negatives of ERK1 and Rac have beendemonstrated, for example, to inhibit expression of a collagenpromoter/reporter gene in TGFβ-stimulated 3T3 fibroblasts. Moreover, instellate liver cells, for instance, evidence has been shown that Raf andthe JNK pathway interact to control cell proliferation and collagenexpression. A number of cytokines, some of which are known to activateSAPKs in cells, have been implicated in cachexia. Accordingly, the novelhuman serine/threonine stress-activated kinase signal transductionmolecule described herein (SEQ ID NO:3), and nucleic acid sequencescoding therefor, e.g. SEQ ID NO: I and SEQ ID NO:2, have significantpotential as specific targets that can be exploited diagnostically andtherapeutically in the control of dysfunctional fibroblasts, includingbut not limited to conditions such as pathological fibrosis, andcachexia as well as other diseases manifested by dysfunctionalfibroblasts.

[0097] The establishment and remodeling of blood vessels is controlledby paracrine signals, many of which are protein ligands that bind andmodulate the activity of transmembrane receptor tyrosine kinases (RTKs).The basic view of RTK signaling has come from studies (performed largelyin fibroblasts) of ligand-dependent autophosphorylation and activationof the branched Ras pathways. The results suggest that most RTKs aresimilarly coupled into the intracellular signal transduction cascade andare capable of inducing cell proliferation. Hanahan, D., SignalingVascular Morphogenesis and Maintenance, Science, 227:48 (1997).Angiogenic response of vascular endothelium, endothelial cellproliferation, is one of the first steps in the angiogenic process whichhas clearly been demonstrated to be induced by hypoxia stress. VEGF,bFGF, and EGF all have been demonstrated to upregulate MAPK in HUVECcells. Accordingly, the novel human serine/threonine stress-activatedkinase signal transduction molecule described herein (SEQ ID NO:3), andnucleic acid sequences coding therefor, e.g. SEQ ID NO:1 and SEQ IDNO:2, have significant potential as specific targets that can beexploited diagnostically and therapeutically to control angiogenesis aswell as other manifestations of dysfunctional fibroblasts.

[0098] Stress-activated protein kinase (SAPK), members of the ERKfamily, are activated in situ by inflammatory stimuli, includingtumour-necrosis factor (TNF). TNF is believed to be responsible for thedevelopment of insulin resistance associated with obesity. Exposure ofcultured cells to TNFα induces insulin resistance which is believed tobe mediated by the Type I TNFα receptor and intracellular signalingmechanisms. In view of the evidence that TNFα is intimately associatedwith the activation of Stress Activated Protein Kinases (SAPKs), thenovel human serine/threonine stress-activated kinase signal transductionmolecule described herein (SEQ ID NO:3), and nucleic acid sequencescoding therefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significantpotential as specific targets that can be exploited diagnostically andtherapeutically to control Diabetes and related disorders.

[0099] The leptin receptor belongs to the cytokine receptor superfamilyof which several members have been shown to feed into SAPK pathways.Intracellular signaling pathways utilized by leptin and the potentialfor regulation of the leptin receptor through “cross-talk” with othersignaling pathways is expected to lead to the design of noveltherapeutic approaches for the treatment of obesity. Accordingly, thenovel human serine/threonine stress-activated kinase signal transductionmolecule described herein (SEQ ID NO:3), and nucleic acid sequencescoding therefor, e.g. SEQ ID NO:1 and SEQ ID NO:2, have significantpotential as specific targets that can be exploited diagnostically andtherapeutically to control Obesity and related disorders.

[0100] Compounds which modulate or inactivate specific signaltransduction molecules integral to specific cytosolic pathways generallyhave significant potential for the ability to modulate or attenuatedownstream physiological responses. Accordingly, the ability to screenfor compounds which modulate the activity of the native humanstress-activated serine/threonine signal transduction kinase moleculedescribed herein is of paramount importance toward the development oftherapeutic agents.

[0101] Functional Constructions:

[0102] The cDNA for the kinase of the present invention has beensubcloned into several expression vectors for the purpose of providingrecombinant protein useful for drug screen assays and for furthertesting the physiological role of the kinase. A dominantnegative/inactive kinase mutant, for example, has been produced whichwill be used to further characterize the physiological role of thekinase. The cDNA has been subcloned into an expression vector in theantisense orientation to provide a tool for producing gene knock-outstudies of gene function. Expression studies are performed using eitherconstitutive or induced expression systems in mammalian cell lines. Theconstructs herein described are example embodiments to be used totransfect mammalian cell lines, e.g., U937 and other well-known celllines, in order to provide recombinant protein suitable for use in drugscreen assays. Constructs are contemplated for over-expressing thewild-type and/or dominant negative kinase of the present invention forcharacterization its physiological role including activation stimuli,identification of signal transduction pathways, identification ofprotein-protein interactions, as well as validation of optimal drugcandidates. Down-regulation of the native signal transduction kinasemolecule described herein is also contemplated using antisenseexpression.

[0103] A constitutive expression construct containing a coding regionfor the novel kinase was produced using pEBVHisA (InvitrogenCorporation). This expression construct is designed to express anN-terminal epitope tagged fusion protein containing the proteinsequence:

[0104] Methionine —(Histidine)₆—Enterokinase protease cleavagesite/Xpress™ antibody epitope—kinase protein (SEQ ID NO:3)

[0105] The SEQ ID NO:1 cDNA was excised from the pT7Blue (Novagen, Inc.)(EXAMPLE I) construct and inserted into the pEBVHisA vector using KpnIand HindIII restriction endonuclease sites at the 5′end and 3′end,respectively.

[0106] Protein expression using the SEQ ID NO:1 cDNA/pEBVHisA constructencodes a His₆-Xpress Ab epitope-Kinase fusion protein. Somecharacteristics of the chimeric molecule include, 482 amino acids,molecular weight 53669.60 daltons, isoelectric point of 5.384, and−15.707 net charge at PH 7.0.

[0107] An inducible expression construct containing a coding region forthe novel kinase was produced using pIND ecdysone inducible expressionvector (Invitrogen Corporation). This expression construct is designedto express an N-terminal epitope tagged fusion protein containing theprotein sequence:

[0108] Methionine—(Histidine)₆—Enterokinase protease cleavagesite/Xpress™ antibody epitope—kinase protein (SEQ ID NO:3)

[0109] The SEQ ID NO:1 cDNA was excised from the pT7Blue (Novagen, Inc.)(EXAMPLE I) construct and inserted into the pIND vector. The H₆-XpressAb epitope-SEQ ID NO:1 insert was excised using ApaLI and XbaIrestriction endonuclease sites at the 5′end and 3′end, respectively. TheApaLI site was filled-in using Klenow DNA polymerase to produce a bluntend. The insert was then ligated into the EcoRV and XbaI restrictionendonuclease sites of the pIND vector.

[0110] A dominant negative/inactive kinase mutant version of the kinasedescribed herein was produced by changing the lysine at SEQ ID NO:3position 53 to an arginine. See FIG. 8. This mutation has been shown toproduce inactive and/or dominant negative kinases for numerous otherserine/threonine kinases. See, e.g., Hanks, S. K., et al. Science,241:42 (1988). The mutation was introduced into the SEQ ID NO:1/pT7Blueconstruct using the Quick Change Site Directed Mutagenesis Kit(Stratagene) and the oligonucleotides shown in FIG. 8 (SEQ ID NO:6 andSEQ ID NO:7). This produced the K53R/pT7Blue construct.

[0111] A constitutive expression construct containing a coding regionfor the novel kinase K53R dominant negative mutant was produced usingpEBVHisA (Invitrogen Corporation). This expression construct is designedto express an N-terminal epitope tagged fusion protein containing thefollowing protein sequence:

[0112] Methionine—(Histidine)₆—Enterokinase protease cleavagesite/Xpress™ antibody epitope—K53R dominant negative mutant kinaseprotein

[0113] The SEQ ID NO:1/K53R cDNA was excised from the K53R/pT7Blueconstruct and inserted into the pEBVHisA vector using KpnI and HindIIIrestriction endonuclease sites at the 5′end and 3′end, respectively.

[0114] Generally Acceptable Vectors:

[0115] In accordance with the present invention, polynucleotidesequences which encode the novel kinase, fragments of the polypeptide,fusion proteins or functional equivalents thereof may be used inrecombinant DNA molecules that direct the expression of the signaltransduction molecule in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequence,may be used to clone and express the novel human signal-transductionkinase. As will be understood by those of skill in the art, it may beadvantageous to produce novel kinase-encoding nucleotide sequencespossessing non-naturally occurring codons.

[0116] Specific initiation signals may also be required for efficienttranslation of a signal- transduction kinase sequence. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherethe novel kinase, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic.

[0117] Cloned signal transduction kinaseDNA obtained through the methodsdescribed herein may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce the kinase polypeptide.Techniques for such manipulations are fully described in Sambrook, J.,et al., Molecular Cloning Second Edition, Cold Spring Harbor Press(1990), and are well known in the art.

[0118] Expression vectors are described herein as DNA sequences for thetranscription of cloned copies of genes and the translation of theirmRNAs in an appropriate host cell. Such vectors can be used to expressnucleic acid sequences in a variety of hosts such as bacteria, bluegreenalgae, plant cells, insect cells, fungal cells, human, and animal cells.Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast, or bacteria-animal cells, or bacteria-fungalcells, or bacteria-invertebrate cells.

[0119] A variety of mammalian expression vectors may be used to expressthe recombinant human kinase molecule disclosed herein in mammaliancells. Commercially available mammalian expression vectors which aresuitable for recombinant expression, include but are not limited to,pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC37565), pLXIN and pSIR (CLONTECH), pIRES-EGFP (CLONTECH). INVITROGENcorporation provides a wide variety of commercially available mammalianexpression vector/systems which can be effectively used with the presentinvention. INVITROGEN, Carlsbad, Calif. See, also, PHARMINGEN products,vectors and systems, San Diego, Calif.

[0120] Baculoviral expression systems may also be used with the presentinvention to produce high yields of biologically active protein. Vectorssuch as the CLONETECH, BacPakTm Baculovirus expression system andprotocols are preferred which are commercially available. CLONTECH, PaloAlto, Calif. Miller, L. K., et al., Curr. Op. Genet. Dev. 3:97 (1993);O'Reilly, D. R., et al., Baculovirus Expression Vectors: A LaboratoryManual, 127. Vectors such as the INVITROGEN, MaxBac™ Baculovirusexpression system, insect cells, and protocols are also preferred whichare commercially available. INVITROGEN, Carlsbad, Calif.

[0121] Example Host Cells:

[0122] Host cells transformed with a nucleotide sequence which encodesthe human kinase of the present invention may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. Particularly preferred embodiments of thepresent invention are host cells transformed with a purifiedpolynucleotide comprising a nucleic acid sequence encoding thepolypeptide having the sequence substantially as depicted in SEQ ID NO:3or a biologically active fragment thereof. Cells of this type orpreparations made from them may be used to screen for pharmacologicallyactive modulators of the novel human signal-transduction kinaseactivity.

[0123] Eukaryotic recombinant host cells are especially preferred.Examples include but are not limited to yeast, mammalian cells includingbut not limited to cell lines of human, bovine, porcine, monkey androdent origin, and insect cells including but not limited to Drosophilaand silkworm derived cell lines. Cell lines derived from mammalianspecies which may be suitable and which are commercially available,include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cellsL-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCCCCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2),C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

[0124] The expression vector may be introduced into host cellsexpressing the novel kinase via any one of a number of techniquesincluding but not limited to transformation, transfection, lipofection,protoplast fusion, and electroporation. Commercially available kitsapplicable for use with the present invention for heterologousexpression, including well- characterized vectors, transfection reagentsand conditions, and cell culture materials are well-established andreadily available. CLONTECH, Palo Alto, Calif.; INVITROGEN, Carlsbad,Calif.; PHARMINGEN, San Diego, Calif.; STRATAGENE, LaJolla, Calif. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine the level of novel kinase proteinproduction. Identification of host cell clones which express the novelkinase may be performed by several means, including but not limited toimmunological reactivity with antibodies described herein, and/or thepresence of host cell-associated specific kinase activity, and/or theability to covalently cross-link specific substrate to the novel kinasewith the bifunctional cross-linking reagent disuccinimidyl suberate orsimilar cross-linking reagents.

[0125] The signal transduction molecule of the present invention mayalso be expressed as a recombinant protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath, J., Protein Exp. Purif.3:263 (1992)), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor XA or enterokinase(Invitrogen, San Diego Calif.) between the purification domain and TMPis useful to facilitate purification.

[0126] Systems such as the CLONTECH, TALON™ nondenaturing proteinpurification kit for purifying 6xHis-tagged proteins under nativeconditions and protocols are preferred which are commercially available.CLONTECH, Palo Alto, CA.

[0127] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a nascent form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, W138,NIH-3T3, HEK293 etc., have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theintroduced, foreign protein.

[0128] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the novel kinase may be transformed using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced sequences. Resistant clumps of stably transformed cells canbe proliferated using tissue culture techniques appropriate to the celltype.

[0129] The human kinase can be produced in the yeast S. cerevisiaefollowing the insertion of the optimal cDNA cistron into expressionvectors designed to direct the intracellular or extracellular expressionof the heterologous protein. In the case of intracellular expression,vectors such as EmBLyex4 or the like are ligated to the beta subunitcistron. See, e.g., Rinas, U., et al., Biotechnology, 8:543 (1990);Horowitz, B., et al., J. Biol. Chem., 265:4189 (1989). For extracellularexpression, the kinase cistron is ligated into yeast expression vectorswhich may employ any of a series of well-characterized secretionsignals. The levels of expressed novel kinase are determined by theassays described herein.

[0130] A variety of protocols for detecting and measuring the expressionof the novel kinase, using either polyclonal or monoclonal antibodiesspecific for the protein are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) andfluorescent activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes may be employed. Well known competitive bindingtechniques may also be employed. See, e.g., Hampton, R., et al. (1990),Serological Methods—a Laboratory Manual, APS Press, St Paul Minn.;Maddox, D. E., et al., J. Exp. Med. 158:1211.

[0131] Yeast 2-Hybrid System:

[0132] In another embodiment of the invention, a nucleic acid sequencewhich encodes a human signal-transduction kinase molecule substantiallyas depicted in SEQ ID NO:3 or a biologically active fragment thereof maybe ligated to a heterologous sequence to encode a fusion protein. Forexample, for screening compounds for modulation of biological activity,it may be useful to encode a chimeric kinase molecule as describedherein for expression in heterologous host cells. Chimeric constructsmay also be used to express a ‘bait’, according to methods well knownusing a yeast two-hybrid system, to identify accessory native peptidesthat may be associated with the novel human signal-transduction kinasemolecule described herein. Fields, S., et al., Trends Genet., 10:286(1994); Allen, J. B., et al., TIBS, 20:511 (1995). A yeast two-hybridsystem has been described wherein protein:protein interactions can bedetected using a yeast-based genetic assay via reconstitution oftranscriptional activators. Fields, S., Song, O., Nature 340:245 (1989).The two-hybrid system used the ability of a pair of interacting proteinsto bring a transcription activation domain into close proximity with aDNA-binding site that regulates the expression of an adjacent reportergene. Commercially available systems such as the CLONTECH, Matchmaker™systems and protocols may be used with the present invention. CLONTECH,Palo Alto, Calif. See also, Mendelsohn, A. R., Brent, R., Curr. Op.Biotech., 5:482 (1994); Phizicky. E. M., Fields, S., MicrobiologicalRev., 59(1): 94 (1995); Yang, M., et al., Nucleic Acids Res., 23(7):1152 (1995); Fields, S., Sternglanz, R., TIG, 10(8): 286 (1994); andU.S. Pat. No. 5,283,173, System to Detect Protein-Protein Interactions,and U.S. Pat. No. 5,468,614, which are incorporated herein by reference.

[0133] Antibodies:

[0134] Monospecific antibodies to the signal transduction kinasepolypeptide of the present invention are purified from mammalianantisera containing antibodies reactive against the polypeptide or areprepared as monoclonal antibodies reactive with signal transductionkinase polypeptide using the technique of Kohler and Milstein, Nature,256:495 (1975). Mono-specific antibody as used herein is defined as asingle antibody species or multiple antibody species with homogenousbinding characteristics for the novel signal transduction kinase.Homogenous binding as used herein refers to the ability of the antibodyspecies to bind to a specific antigen or epitope, such as thoseassociated with the novel signal transduction kinase, as described.Novel signal transduction kinase specific antibodies are raised byimmunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with rabbits being preferred, with an appropriateconcentration of the human signal transduction kinase either with orwithout an immune adjuvant.

[0135] Preimmune serum is collected prior to the first immunization.Each animal receives between about 0.1 mg and about 1000 mg of signaltransduction kinase polypeptide associated with an acceptable immuneadjuvant. Such acceptable adjuvants include, but are not limited to,Freund's complete, Freund's incomplete, alum-precipitate, water in oilemulsion containing Corynebacterium parvum and tRNA. The initialimmunization consists of signal transduction kinase polypeptide in,preferably, Freund's complete adjuvant at multiple sites eithersubcutaneously (SC), intraperitoneally (IP) or both. Each animal is bledat regular intervals, preferably weekly, to determine antibody titer.The animals may or may not receive booster injections following theinitial immunization. Those animals receiving booster injections aregenerally given an equal amount of the antigen in Freund's incompleteadjuvant by the same route. Booster injections are given at about threeweek intervals until maximal titers are obtained. At about 7 days aftereach booster immunization or about weekly after a single immunization,the animals are bled, the serum collected, and aliquots are stored atabout −20° C.

[0136] Monoclonal antibodies (mAb) reactive with the signal transductionkinase polypeptide are prepared by immunizing inbred mice, preferablyBalb/c, with the signal transduction kinase polypeptide. The mice areimmunized by the IP or SC route with about 0.1 mg to about 10 mg,preferably about 1 mg, of signal transduction kinase polypeptide inabout 0.5 ml buffer or saline incorporated in an equal volume of anacceptable adjuvant, as discussed above. Freund's complete adjuvant ispreferred. The mice receive an initial immunization on day 0 and arerested for about 3 to about 30 weeks. Immunized mice are given one ormore booster immunizations of about 0.1 to about 10 mg of signaltransduction kinase polypeptide in a buffer solution such as phosphatebuffered saline by the intravenous (IV) route. Lymphocytes, fromantibody positive mice, preferably splenic lymphocytes, are obtained byremoving spleens from immunized mice by standard procedures known in theart. Hybridoma cells are produced by mixing the splenic lymphocytes withan appropriate fusion partner, preferably myeloma cells, underconditions which will allow the formation of stable hybridomas. Fusionpartners may include, but are not limited to: mouse myelomas P3/NS1/Ag4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibodyproducing cells and myeloma cells are fused in polyethylene glycol,about 1000 molecular weight, at concentrations from about 30% to about50%. Fused hybridoma cells are selected by growth in hypoxanthine,thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium(DMEM) by procedures known in the art. Supernatant fluids are collectedfrom growth positive wells on about days 14, 18, and 21 and are screenedfor antibody production by an immunoassay such as solid phaseimmunoradioassay (SPIRA) using the human signal transduction kinasepolypeptide as the antigen. The culture fluids are also tested in theOuchterlony precipitation assay to determine the isotype of the mAb.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, Soft Agar Techniques, inTissue Culture Methods and Applications, Kruse and Paterson, Eds.,Academic Press, 1973.

[0137] Monoclonal antibodies are produced in vivo by injection ofpristane primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

[0138] In vitro production of the anti- human kinase polypeptide mAb iscarried out by growing the hydridoma in DMEM containing about 2% fetalcalf serum to obtain sufficient quantities of the specific mAb. The mAbare purified by techniques known in the art.

[0139] Diagnostic Assays:

[0140] Antibody titers of ascites or hybridoma culture fluids aredetermined by various serological or immunological assays which include,but are not limited to, precipitation, passive agglutination,enzyme-linked immunosorbent antibody (ELISA) technique andradioimmunoassay (RIA) techniques. Similar diagnostic assays are used todetect the presence of the novel signal transduction kinase polypeptidein body fluids or tissue and cell extracts.

[0141] Diagnostic assays using the human signal-transduction kinasepolypeptide specific antibodies are useful for the diagnosis ofconditions, disorders or diseases characterized by abnormal expressionof the signal-transduction kinase polypeptide or expression of genesassociated with abnormal cell growth. Diagnostic assays for thesignal-transduction kinase polypeptide of this invention include methodsutilizing the antibody and a label to detect the human kinasepolypeptide in human body fluids, cells, tissues or sections or extractsof such tissues. The polypeptides and antibodies of the presentinvention may be used with or without modification. Frequently, thepolypeptides and antibodies will be labeled by joining them, eithercovalently or noncovalently, with a reporter molecule, a myriad of whichare well-known to those skilled in the art.

[0142] A variety of protocols for measuring the kinase polypeptide,using either polyclonal or monoclonal antibodies specific for therespective protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson the human signal-transduction kinase polypeptide is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D. E. et al., J. Exp. Med. 158:1211(1983); Sites, D. P., et al., Basic and Clinical Immunology, Ch.22, 4thEd., Lange Medical Publications, Los Altos, Calif. (1982); U.S. Pat. No.3,654,090, No. 3,850,752; and No. 4,016,043.

[0143] In order to provide a basis for the diagnosis of disease, normalor standard values for the human signal-transduction kinase polypeptideexpression must be established. This is accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with antibody to the human kinase polypeptide under conditionssuitable for complex formation which are well known in the art. Theamount of standard complex formation may be quantified by comparing itwith a dilution series of positive controls where a known amount ofantibody is combined with known concentrations of purified humansignal-transduction kinase polypeptide. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by a disorder or disease related tothe human kinase polypeptide expression. Deviation between standard andsubject values establishes the presence of the disease state.

[0144] Kits containing human stress-activated signal-transduction kinasenucleic acid, antibodies to a kinase polpeptide, or protein may beprepared. Such kits are used to detect heterologous nucleic acid whichhybridizes to kinase nucleic acid, or to detect the presence of proteinor peptide fragments in a sample. Such characterization is useful for avariety of purposes including, but not limited to, forensic analyses andepidemiological studies.

[0145] The DNA molecules, RNA molecules, recombinant protein andantibodies of the present invention may be used to screen and measurelevels of the novel kinase DNA, RNA or protein. The recombinantproteins, DNA molecules, RNA molecules and antibodies lend themselves tothe formulation of kits suitable for the detection and typing of thehuman signal-transduction kinase. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier would further comprise reagents such asrecombinant human kinase or anti-kinase antibodies suitable fordetecting the novel kinase. The carrier may also contain a means fordetection such as labeled antigen or enzyme substrates or the like.

[0146] Polynucleotide sequences which encode the novel kinase may beused for the diagnosis of conditions or diseases with which theexpression of the novel human stress-activated kinase is associated. Forexample, polynucleotide sequences encoding the signal-transductionmolecule may be used in hybridization or PCR assays of fluids or tissuesfrom biopsies to detect expression of the kinase. The form of suchqualitative or quantitative methods may include Southern or northernanalysis, dot blot or other membrane-based technologies; PCRtechnologies; dip stick, pin, chip and ELISA technologies. All of thesetechniques are well known in the art and are the basis of manycommercially available diagnostic kits. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regime inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient. Once disease is established, a therapeutic agent isadministered and a treatment profile is generated. Such assays may berepeated on a regular basis to evaluate whether the values in theprofile progress toward or return to the normal or standard pattern.Successive treatment profiles may be used to show the efficacy oftreatment over a period of several days or several months.

[0147] Polynucleotide sequences which encode the novel kinase may alsobe employed in analyses to map chromosomal locations, e.g., screeningfor functional association with disease markers. Moreover the sequencesdescribed herein are contemplated for use to identify human sequencepolymorphisms and possible association with disease as well as analysesto select optimal sequence from among possible polymorphic sequences forthe design of compounds to modulate the biological activity. Furthermorethe sequences are contemplated as screening tools for use in theidentification of appropriate human subjects and patients fortherapeutic clinical trials.

[0148] Purification Via Affinity Columns:

[0149] It is readily apparent to those skilled in the art that methodsfor producing antibodies may be utilized to produce antibodies specificfor the human kinase polypeptide fragments, or the full-length nascenthuman kinase polypeptide. Specifically, it is readily apparent to thoseskilled in the art that antibodies may be generated which are specificfor the fully functional receptor or fragments thereof.

[0150] Kinase polypeptide antibody affinity columns are made by addingthe antibodies to Affigel-10 (Biorad), a gel support which is activatedwith N hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) with appropriatedetergent and the cell culture supernatants or cell extracts containinghuman signal-transduction kinase polypeptide made using appropriatemembrane solubilizing detergents are slowly passed through the column.The column is then washed with phosphate buffered saline/detergent untilthe optical density falls to background, then the protein is eluted with0.23M glycine-HCl (pH 2.6)/detergent. The purified humansignal-transduction kinase polypeptide is then dialyzed againstphosphate buffered saline/detergent.

[0151] Recombinant kinase molecules can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full length nascent human kinasepolypeptide, or polypeptide fragments of the kinase molecule.

[0152] Human kinase polypeptides described herein may be used toaffinity purify biological effectors from native biological materials,e.g. disease tissue. Affinity chromatography techniques are well knownto those skilled in the art. A human signal-transduction kinase peptidedescribed herein or an effective fragment thereof, is fixed to a solidmatrix, e.g. CNBr activated Sepharose according to the protocol of thesupplier (Pharmacia, Piscataway, N.J.), and a homogenized/bufferedcellular solution containing a potential molecule of interest is passedthrough the column. After washing, the column retains only thebiological effector which is subsequently eluted, e.g., using 0.5Macetic acid or a NaCl gradient.

[0153] Antisense Molecules:

[0154] The cDNA sequence SEQ ID NO:1 provided herein, may be used inanother embodiment of the invention to study the physiological relevanceof the novel human signal- transduction kinase in cells, especiallycells of hematopoietic origin, by knocking out the endogenous gene byuse of anti-sense constructs.

[0155] To enable methods of down-regulating expression of the novelhuman kinase of the present invention in mammalian cells, an exampleantisense expression construct containing the complement DNA sequence(5′→3′ is shown in FIG. 7 (SEQ ID NO:5)) to the novel kinase cDNA wasproduced using the pREP10 vector (Invitrogen Corporation). The SEQ IDNO:1 cDNA was excised from the pT7Blue (Novagen, Inc.) construct andinserted into the pREP10 vector using KpnI and HindIII restrictionendonuclease sites at the 5′end and 3′end, respectively. This places theSEQ ID NO:1 cDNA in an anti-sense orientation with respect to thepromoter and is expected to express an antisense mRNA transcript.Transcripts are expected to inhibit translation of the wild-type kinasemRNA in cells transfected with this construct. Oligomers of 12-21nucleotides are most preferred in the practice of the present invention,particularly oligomers of 12-18 nucleotides. Transcript are, inprinciple, effective for inhibiting translation of the transcript, andcapable of inducing the effects herein described. Translation is mosteffectively inhibited by blocking the mRNA at a site at or near theinitiation codon. Thus, oligonucleotides complementary to the5′-terminal region of the human kinase mRNA transcript are preferred.Secondary or tertiary structure which might interfere with hybridizationis minimal in this region. Moreover, sequences that are too distant inthe 3′ direction from the initiation site can be less effective inhybridizing the mRNA transcripts because of a “read-through” phenomenonwhereby the ribosome appears to unravel the antisense/sense duplex topermit translation of the message. Oligonucleotides which arecomplementary to and hybridizable with any portion of the novel humansignal- transduction kinase mRNA are contemplated for therapeutic use

[0156] U.S. Pat. No. 5,639,595, Identification of Novel Drugs andReagents, issued Jun. 17, 1997, wherein methods of identifyingoligonucleotide sequences that display in vivo activity are thoroughlydescribed, is herein incorporated by reference. Expression vectorscontaining random oligonucleotide sequences derived from previouslyknown polynucleotides are transformed into cells. The cells are thenassayed for a phenotype resulting from the desired activity of theoligonucleotide. Once cells with the desired phenotype have beenidentified, the sequence of the oligonucleotide having the desiredactivity can be identified. Identification may be accomplished byrecovering the vector or by polymerase chain reaction (PCR)amplification and sequencing the region containing the inserted nucleicacid material.

[0157] Nucleotide sequences that are complementary to the novelsignal-transduction kinase polypeptide encoding polynucleotide sequencecan be synthesized for antisense therapy. These antisense molecules maybe DNA, stable derivatives of DNA such as phosphorothioates ormethylphosphonates, RNA, stable derivatives of RNA such as2′-O-alkylRNA, or other oligonucleotide mimetics. U.S. Pat. No.5,652,355, Hybrid Oligonucleotide Phosphorothioates, issued Jul. 29,1997, and U.S. Pat. No. 5,652,356, Inverted Chimeric and HybridOligonucleotides, issued Jul. 29, 1997, which describe the synthesis andeffect of physiologically-stable antisense molecules, are incorporatedby reference. Signal-transduction kinase antisense molecules may beintroduced into cells by microinjection, liposome encapsulation or byexpression from vectors harboring the antisense sequence. Antisensetherapy may be particularly useful for the treatment of diseases whereit is beneficial to reduce the signal-transduction kinase activity.

[0158] Gene Therapy:

[0159] A human signal-transduction kinase polypeptide described hereinmay administered to a subject via gene therapy. Moreover, a polypeptideof the present invention may be delivered to the cells of target organsin this manner. Conversely, signal-transduction kinase polypeptideantisense gene therapy may be used to reduce the expression of thepolypeptide in the cells of target organs. The human signal-transductionkinase polypeptide coding region can be ligated into viral vectors whichmediate transfer of the kinase polypeptide DNA by infection of recipienthost cells. Suitable viral vectors include retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, polio virus andthe like. See, e.g., U.S. Pat. No. 5,624,820, Episomal Expression Vectorfor Human Gene Therapy, issued Apr. 29, 1997. Nucleic acid codingregions of the present invention are incorporated into effectiveeukaryotic expression vectors, which are directly administered orintroduced into somatic cells for gene therapy (a nucleic acid fragmentcomprising a coding region, preferably mRNA transcripts, may also beadministered directly or introduced into somatic cells). See, e.g., U.S.Pat. No. 5,589,466, issued Dec. 31, 1996. Such nucleic acids and vectorsmay remain episomal or may be incorporated into the host chromosomal DNAas a provirus or portion thereof that includes the gene fusion andappropriate eukaryotic transcription and translation signals, i.e., aneffectively positioned RNA polymerase promoter 5′ to the transcriptionalstart site and ATG translation initiation codon of the gene fusion aswell as termination codon(s) and transcript polyadenylation signalseffectively positioned 3′ to the coding region. Alternatively, the humansignal-transduction kinase polypeptide DNA can be transferred into cellsfor gene therapy by non-viral techniques including receptor-mediatedtargeted DNA transfer using ligand-DNA conjugates oradenovirus-ligand-DNA conjugates, lipofection membrane fusion or directmicroinjection. These procedures and variations thereof are suitable forex vivo, as well as in vivo human signal-transduction kinase polypeptidegene therapy according to established methods in this art.

[0160] Screening Assays:

[0161] The present invention is also directed to methods for screeningfor compounds which modulate the expression of DNA or RNA encoding thenovel human kinase polypeptide, as well as the function of the humansignal-transduction kinase polypeptide in vivo. Compounds which modulatethese activities may be DNA, RNA, peptides, proteins, ornon-proteinaceous organic molecules. Compounds may modulate byincreasing or attenuating the expression of DNA or RNA encoding thehuman signal-transduction kinase polypeptide, or the function thereof.Compounds that modulate the expression of DNA or RNA encoding the humansignal-transduction kinase polypeptide or the function of thepolypeptide may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample.

[0162] The human signal-transduction kinase described herein, itsimmunogenic fragments or oligopeptides can be used for screeningtherapeutic compounds in any of a variety of drug screening techniques.The fragment employed in such a test may be free in solution, affixed toa solid support, borne on a cell surface, or located intracellularly.The abolition of activity or the formation of binding complexes, betweenthe human signal-transduction kinase polypeptide and the agent beingtested, may be measured. Accordingly, the present invention provides amethod for screening a plurality of compounds for specific bindingaffinity with the human signal-transduction kinase polypeptide or afragment thereof, comprising providing a plurality of compounds;combining the human signal-transduction kinase polypeptide of thepresent invention or a fragment thereof with each of a plurality ofcompounds for a time sufficient to allow binding under suitableconditions; and detecting binding of the kinase polypeptide, or fragmentthereof, to each of the plurality of compounds, thereby identifying thecompounds which specifically bind the human signal-transduction kinasepolypeptide.

[0163] Methods of identifying compounds that modulate the activity of ahuman signal-transduction kinase polypeptide are generally preferred,which comprise combining a candidate compound modulator of a humansignal-transduction kinase activity with a polypeptide of a humansignal-transduction kinase having the sequence substantially as depictedin SEQ ID NO:3, and measuring an effect of the candidate compoundmodulator on the kinase activity.

[0164] Methods of identifying compounds that modulate the activity of ahuman signal- transduction kinase, are also preferred which comprisecombining a candidate compound modulator of a human signal-transductionkinase activity with a host-cell expressing the polypeptide of a humansignal-transduction kinase molecule having the sequence substantially asdepicted in SEQ ID NO:3, and measuring an effect of the candidatecompound modulator on the kinase activity. Preferred cellular assays offor inhibitors of the kinase fall into two general categories: 1) directmeasurement of the kinase activity, and 2) measurement of a downstreamevents in the signaling cascade. These methods can employ the endogenouskinase, or the overexpressed recombinant kinase.

[0165] In order to measure the cellular activity of the kinase, thesource may be a whole cell lysate, prepared by one to three freeze-thawcycles in the presence of standard protease inhibitors. Alternatively,the kinase may be partially or completely purified by standard proteinpurification methods. Finally, the kinase may be purified by affinitychromatography using specific antibody for the C terminal regulatorydomain described herein or by ligands specific for the epitope tagengineered into the recombinant kinase moreover described herein. Thekinase preparation may then be assayed for activity as described, forexample, in Example II.

[0166] A filter assay based on the protocol of Reuter et al. (1995) isalso used to screen for compounds which modulate the activity of thenovel kinase described herein: Starting with MBP coated 96-wellFlashPlates®) (NEN™ Life Science Products) reaction buffer (3× kinasereaction buffer (KRB) contains: 60 mM HEPES (pH 7.5), 30 mM magnesiumacetate, 0.15 mM ATP, 3 mM DTT, 0.03 mM sodium orthovanadate) is added,0.25 μCi [γ ³³P]-ATP at a concentration no greater than 1 μg/ml,(determined by titration of individual enzyme preparations for aconcentration that allows kinetic determinations over a 1 hour timecourse of the human kinase) of the human kinase are added to each welland incubated 1 hour at 30° C. in the presence or absence of 10 μM testcompound. Total reaction volume is 100 μL. The reaction is stopped bythe addition of EDTA (pH 7.0) to a final concentration of 80 mM. Thesamples are centrifuged and 50 μL of the supernatant spotted on p81cation-exchange filter paper (Whatman, No. 3698 915). The filters arethen washed 3 times in 200 mL of 180 mM H₃PO₄ (5-10 min each), and oncein 200 mL of 96% ethanol. After air drying the filters, radioactivity isdetermined by Cerenkov counting in a scintillation counter. Compoundswhich inhibit kinase activity >50 percent at 10 μM are indicated bya >50% reduction in scintillation counts. Specificity and selectivitystudies is determined by titration of inhibitory compounds to determinethe IC₅₀ (or other standard quantitation well known in the art forcomparison) and by the substitution of other kinases in the assay. Forexample, determination of relative inhibitory activity of the kinase incomparison to recombinant SOK-1, expressed and isolated in a similarmanner, assayed under similar conditions, provides selectivity data.Reuter, C. W. M., Catling, A. D. and Weber, M. J., Immune Complex KinaseAssays for Mitogen-Activated Protein Kinase and MEK, Methods InEnzymology, 255:245 (1995).

[0167] To evaluate the ability of a candidate agent to inhibit humantumor growth, human tumor cells are injected into SCID mice (severecombined immunodeficiency) to form palpable tumor masses. The effects ofan candidate agent in inhibiting tumor growth can be determined asfollows. Approximately 1×10⁷ cells of the CCL 221 cell line (ATCC,Rockville, Md.), a human ras-dependent colon adenocarcinoma cell line,is suspended in 100 μL DMEM and injected subcutaneously into SCID mice,such that two tumors per mouse are formed. SCID mice receive CCL 221cells and the tumors are grown for 7 days without treatment; on the 7thday (Day 0) tumor maximal diameters and animal weights are recorded andthe mean tumor size for the mice is determined. On Day 1 (eight daysfollowing tumor cell injection), treatment of the mice with candidateagent or vehicle alone is begun. One group of the mice (controls) areinjected intraperitoneally with 0.2 ml of vehicle and a second group ofmice received agent by intraperitoneal injection. Various doses of agentcan be tested in separate groups of mice. On Day 7 and Day 14, animalweight and maximal tumor diameter is measured. Average maximal tumorsize for each group on Day 0, Day 7, and Day 14 are compared. One highdose animal was followed for an additional to determined whether theagent produces a dose-dependent inhibition of tumor growth. Toxicityeffects can be examined by tracking mice weight and by harvesting lungs,livers, and spleens of the animals for histological staining.

[0168] Compounds which are identified generally according to methodsdescribed and referenced herein that modulate the activity of a humansignal-transduction kinase comprised of the sequence substantially asdepicted in SEQ ID NO:3 are especially preferred embodiments of thepresent invention.

[0169] An especially preferred embodiment of the present invention is amethod for treatment of a patient in need of such treatment for acondition which is mediated by the human signal-transduction kinasedescribed herein comprising administration of a therapeuticallyeffective amount of a human signal-transduction kinase modulatingcompound.

[0170] PCR Diagnostics:

[0171] The nucleic acid sequence, oligonucleotides, fragments, portionsor antisense molecules thereof, may be used in diagnostic assays of bodyfluids or biopsied tissues to detect the expression level of the novelhuman signal-transduction kinase molecule. For example, sequencesdesigned from the cDNA sequence SEQ ID NO:1 or sequences comprised inSEQ ID NO:2 can be used to detect the presence of the mRNA transcriptsin a patient or to monitor the modulation of transcripts duringtreatment.

[0172] One method for amplification of target nucleic acids, or forlater analysis by hybridization assays, is known as the polymerase chainreaction (“PCR”) or PCR technique. The PCR technique can be applied todetect sequences of the invention in suspected samples usingoligonucleotide primers spaced apart from each other and based on thegenetic sequence, e.g., SEQ ID NO:1, set forth herein. The primers arecomplementary to opposite strands of a double stranded DNA molecule andare typically separated by from about 50 to 450 nucleotides or more(usually not more than 2000 nucleotides). This method entails preparingthe specific oligonucleotide primers followed by repeated cycles oftarget DNA denaturation, primer binding, and extension with a DNApolymerase to obtain DNA fragments of the expected length based on theprimer spacing. One example embodiment of the present invention is adiagnostic composition for the identification of a polynucleotidesequence comprising the sequence substantially as depicted in SEQ IDNO:2 comprising the PCR primers substantially as depicted in SEQ ID NO:8and SEQ ID NO:9. The degree of amplification of a target sequence iscontrolled by the number of cycles that are performed and istheoretically calculated by the simple formula 2n where n is the numberof cycles. See, e.g., Perkin Elmer, PCR Bibliography, Roche MolecularSystems, Branchburg, N.J.; CLONTECH products, Palo Alto, Calif.; U.S.Pat. No. 5,629,158, Solid Phase Diagnosis of Medical Conditions, issuedMay 13, 1997.

[0173] Compositions:

[0174] Pharmaceutically useful compositions comprising the novel humankinase polypeptide DNA, human kinase polypeptide RNA, antisensesequences, or the human kinase polypeptide, or variants and analogswhich have the human kinase activity or otherwise modulate its activity,may be formulated according to known methods such as by the admixture ofa pharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington 's PharmaceuticalSciences (Maack Publishing Co, Easton, Pa.). To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the protein, DNA, RNA,or modulator.

[0175] Therapeutic or diagnostic compositions of the invention areadministered to an individual in amounts sufficient to treat or diagnosehuman signal-transduction kinase polypeptide related disorders. Theeffective amount may vary according to a variety of factors such as theindividual's condition, weight, sex and age. Other factors include themode of administration.

[0176] The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

[0177] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose is well within the capability ofthose skilled in the art. The therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans. A therapeuticallyeffective dose refers to that amount of protein or its antibodies,antagonists, or inhibitors which ameliorate the symptoms or condition.The exact dosage is chosen by the individual physician in view of thepatient to be treated.

[0178] Compounds identified according to the methods disclosed hereinmay be used alone at appropriate dosages defined by routine testing inorder to obtain optimal modulation of a signal-transduction kinase, orits activity while minimizing any potential toxicity. In addition,co-administration or sequential administration of other agents may bedesirable.

[0179] The pharmaceutical compositions may be provided to the individualby a variety of routes such as subcutaneous, topical, oral andintramuscular. Administration of pharmaceutical compositions isaccomplished orally or parenterally. Methods of parenteral deliveryinclude topical, intra-arterial (directly to the tissue), intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration. The presentinvention also has the objective of providing suitable topical, oral,systemic and parenteral pharmaceutical formulations for use in the novelmethods of treatment of the present invention. The compositionscontaining compounds identified according to this invention as theactive ingredient for use in the modulation of signal- transductionkinase can be administered in a wide variety of therapeutic dosage formsin conventional vehicles for administration. For example, the compoundscan be administered in such oral dosage forms as tablets, capsules (eachincluding timed release and sustained release formulations), pills,powders, granules, elixirs, tinctures, solutions, suspensions, syrupsand emulsions, or by injection. Likewise, they may also be administeredin intravenous (both bolus and infusion), intraperitoneal, subcutaneous,topical with or without occlusion, or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compound desired can beemployed as a signal-transduction kinase modulating agent.

[0180] The daily dosage of the products may be varied over a wide rangefrom 0.01 to 1,000 mg per adult human/per day. For oral administration,the compositions are preferably provided in the form of scored orunscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0,15.0, 25.0, and 50.0 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Aneffective amount of the drug is ordinarily supplied at a dosage level offrom about 0.0001 mg/kg to about 100 mg/kg of body weight per day. Therange is more particularly from about 0.001 mg/kg to 10 mg/kg of bodyweight per day. Even more particularly, the range varies from about 0.05to about 1 mg/kg. Of course the dosage level will vary depending uponthe potency of the particular compound. Certain compounds will be morepotent than others. In addition, the dosage level will vary dependingupon the bioavailability of the compound. The more bioavailable andpotent the compound, the less compound will need to be administeredthrough any delivery route, including but not limited to oral delivery.The dosages of the human signal-transduction kinase modulators areadjusted when combined to achieve desired effects. On the other hand,dosages of these various agents may be independently optimized andcombined to achieve a synergistic result wherein the pathology isreduced more than it would be if either agent were used alone. Thoseskilled in the art will employ different formulations for nucleotidesthan for proteins or their inhibitors. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells andconditions.

EXAMPLES Example I

[0181] Sequence Construction, Cloning, Expression and Purification:

[0182] The novel human kinase described herein was identified after theassembly of expressed sequence tags (ESTs) from the Incyte LIFSEQdatabase. Initial identification of these ESTs was performed by basiclocal alignment search tool (BLAST) analysis of the database using thekinase subdomain VIB sequence HRDLKPENILLD previously described. Thishighly conserved sequence tends to be specific for serine/threoninekinases. Altschul, Stephen F., et al., Basic Local Alignment SearchTool, J. Mol. Biol., 215:403 (1990); Altschul, Stephen F., et al., AProtein Alignment Scoring System Sensitive at all EvolutionaryDistances, J. Mol. Evol., 36:290 (1993); Altschul, Stephen F., et al.,Issues in Searching Molecular Sequence Databases, Nature Genetics, 6:119(1994).

[0183] Oligonucleotide primers, sense 5′GAGCGCCATGGCTCACTCC3′ (SEQ IDNO:8) and antisense 5′GGAGTCAGCGAAGGCTCTCG3′ (SEQ ID NO:9), weredesigned based on the predicted sequence to the novel human kinase andwere used to amplify nucleic acid sequence pertaining to the novel humankinase from human brain cDNA (Clontech) and human U937 cell cDNA usingTaq polymerase. Clones were sequenced (ABI PRISM™ Dye Terminator Cyclesequencing on ABI PRISM™377 automated sequencer). This yielded a singlecDNA species of the sequence SEQ ID NO:1, validating the electronicassembly of the cDNA. The PCR products were ligated into the pT7Bluevector (Novagen, Inc.) and used to transform NovaBlue competent cells(Novagen, Inc.). Plasmids were prepared from positive clones forsequencing. A clone was found to have sequence identical to theelectronic contig for the new kinase molecule, nucleic acid SEQ ID NO:1.The 2322 basepair (bp) sequence was found to contain a 1293 bp openreading frame (ORF) with a Kozak initiation sequence at the startmethionine. Translation of the ORF resulted in a 431 amino acid proteinwhich contains all 11 homologous domains found in eukaryotic proteinkinases. The novel kinase has 69.7 percent identity to the Ste20/oxidantstress response kinase-1 (SOK-1) described by Pombo et al. (1996). Theprotein also has a predicted molecular weight of 47,919 daltons, anisolectric point of 5.24 and a net charge of -12.68.

[0184] The pT7Blue plasmid containing SEQ ID NO:1 was gel purified,restriction digested with EcoRI/Sal I, and ligated into the multiplecloning site of the GST gene fusion vector pGEX-SX-2 (Pharmacia Biotech,Inc.). The resultant construct was used to transform NovaBlue competentE. coli cells for maintenance and BL21 competent cells (Novagen, Inc.)for protein expression.

[0185] GST fusion clones were screened for the incorporation of thecorrect sequence. A single BL21 clone was isolated for proteinexpression. This clone was grown overnight in 10 mL Lennox L broth (LBbroth) and seeded into 1 liter LB broth and grown at 37° C. withagitation to an A₆₀₀ of 1.0-2.0. Expression of the GST/novel kinasefusion protein was induced by adding 100 μM isopropylthio-β-galactoside,incubation for 2 more hours. Following incubation, the culture wascentrifuged 1500× g, 10 min, 4° C. and resuspended in 50 mL phosphatebuffered saline (PBS) containing Complete™ Protease Inhibitor Cocktail(Boehringer Mannheim GmbH). Cells were then lysed by sonication on ice.Triton X-100 was added to the sonicate to a final concentration of 1% toaid in the solubilization of the fusion protein. Cellular debris wasremoved by centrifugation (12,000× g, 4° C.).

[0186] The kinase molecule was purified by the addition of GlutathioneSepharose 4B beads (Pharmacia) at a concentration of 1 ml of a 50%slurry in PBS per 50 mL of lysate. The suspension was incubated for 30minutes at room temperature with gentle agitation, centrifuged (500× g,5 min, 4° C.) and washed three times with PBS containing proteaseinhibitors. Finally, the beads were sedimented by centrifugation,resuspended in 1 mL elution buffer (10 mM reduced glutathione in 50 mMTris-HCl, pH 8.0 with protease inhibitors) and incubated 10 minutes atroom temperature to remove the fusion protein from the beads. Beads werespun out (500× g, 5 min) and the GST/kinase fusion was stored inaliquots at −20° C. until needed.

Example II

[0187] Assay for Human Kinase Activity:

[0188] The recombinant, Glutathionine S-transferase (GST)/novel humankinase fusion was constructed by digesting a pT7Blue vector (Novagen,Inc.) containing SEQ ID NO:1 with EcoRI/Sal I, and subsequent ligationinto the multiple cloning site of the GST gene fusion vector pGEX-5X-2(Pharmacia Biotech, Inc.). The resultant construct was used to transformBL21 competent cells (Novagen, Inc.) for protein expression.

[0189] Recombinant, purified GST/kinase (10 μL) was added to 20 μgmyelin basic protein (MBP) in 10 μL of a 3× kinase reaction buffer (KRB)containing: 60 mM HEPES (pH 7.5), 30 mM magnesium acetate, 0.15 mM ATP,3 mM DTT, 0.03 mM sodium orthovanadate. The reaction was started by theaddition of 5 μCi [γ-³²P] ATP (10 μL). Samples were incubated for 5minutes at 30° C. and the reaction was stopped by addition of 4× Laemmlisample buffer. Proteins were separated on 12% Tris/glycine SDS gels,stained with Coomassie blue, dried and exposed to autoradiograph film.

[0190] Results of an autoradiographic assay for the kinase activity ofthe novel human kinase polypeptide described herein is shown in FIG. 6.Lane 1: Novel Kinase+MBP+[γ-³²P] ATP. Lane 2: Novel Kinase+MBP, no[γ-³²P] ATP. Lane 3: MBP+[γ-³²P] ATP, no Novel Kinase. Lane 4: NovelKinase+[γ-³²P] ATP, no MBP. Values at left represent molecular weightstandards in kDa.

[0191] Phosphorylation of MBP (1 8-20kDa) and autophosphorylation of thefusion protein kinase (76.9kDa) are evident in lane 1. No proteinphosphorylation was observed when [γ-³²P] ATP or Novel Kinase wereomitted; lane 2 and 3, respectively. Autophosphorylation increased inthe absence of the nonspecific substrate (lane 4).

Example III

[0192] Production of Anti-Kinase Polyclonal Antibodies:

[0193] Antigenic peptide fragments were identified within theN-terminal, c-terminal and central regions of the novel human kinaseutilizing a well established algorithm method developed by Jameson andWolf. The Antigenic Index: A Novel Algorithm for Predicting AntigenicDeterminants, CABIOS, 4:181 (1988). The algorithm carries out six majorsubroutines with the following hierarchy:

[0194] 1) determination of hydrophilicity, Hopp-Woods (1981)

[0195] 2) calculation of surface probability, Emini (1985)

[0196] 3) prediction of backbone or chain flexibility, Karplus-Schultz(1985)

[0197] 4) prediction of secondary structure, Chou-Fasman (1978)

[0198] 5) prediction of secondary structure, Garnier-Robson (1978)

[0199] 6) flexibility parameters and hydropathy/solvent accessibilityfactors are combined to determine the antigenic index

[0200] The antigenic index was plotted for the entire molecule.

[0201] The following peptides were selected for synthesis and antibodyproduction:

[0202] 1) FKGIDNRTQK residues 39-48 (N-terminal) of SEQ ID NO:3

[0203] 2) DRNKMKDIPKRP residues 348-359 (C-terminal) of SEQ ID NO:3

[0204] 3) NNPPTLEGNYSKPL residues 234-247 (central) of SEQ ID NO:3

[0205] These peptides were conjugated to keyhole limpet hemocyanin usingthe IMJECT® Immunogen EDC Conjugation Kit (Pierce). Each conjugatedpeptide was used to immunize two rabbits according to standard protocols(Harlow and Lane, 1988).

[0206] A 165 amino acid peptide from a likely non-catalytic region ofthe kinase (SEQ ID NO:3 residues 267-432) was PCR cloned and expressedin bacteria from pGEX-5X (Pharmacia) as a GST fusion protein. Thepurified, unconjugated peptide was used to immunize rabbits as describedsupra. Chou, P. Y. and Fasman, G. D., (1978) Prediction of the secondarystructure of proteins from their amino acid sequence, Adv. Enzymol,47:45-148; Emini, E. A., Hughes, J., Perlow, D. and Boger, J., (1985)Induction of Hepatitis A Virus-Neutralizing Antibody by a Virus-SpecificSynthetic Peptide, J. Virology, 55:836-839; Gamier, J., Osguthorpe, D.J., and Robson, B., (1978) Analysis of the accuracy and implications ofsimple method for predicting the secondary structure of globularproteins, J.Mol. Biol., 120:97-120; Harlow, E. and Lane, D., (1988)Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Hopp, T. P. and Woods, K. R., (1981)Prediction of Protein Antigenic Determinants from Amino Acid Sequences,Proc. Natl. Acad. Sci., 78:3824-3828; Jameson, B. A., and Wolf, H.,(1988) The antigenic index: a novel algorithm for predicting antigenicdeterminants, CABIOS 4:181-186; Karplus, P. A. and Schultz, G. E.,(1985) Prediction of chain flexibility in proteins, Naturwissenschaften,72:212-213.

Example IV

[0207] Immunoprecipitation:

[0208] Immunoprecipitation of the human kinase molecule described hereinis performed substantially according to the method described by Suchard,S. J., et al. J. Immunol., 158:4961 (1997). Cell lysates are combinedwith 1 μg of either anti-enterokinase protease cleavage site/Xpress™antibody (Invitrogen Corp.) for the recombinant kinase described hereinor peptide-specific polyclonal antibody against the native kinasedescribed herein. Rabbit IgG is used as a control. Samples are incubatedat 4° C.≧2 hours with rotation. Immunocomplexes are incubated withprotein A Sepharose (Pharmacia) for 2 hours at 4° C. with rotation. Thebeads are washed in buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl,1 mM Na₃VO₄, 1% Triton X-100, and Complete™ Protease Inhibitor Cocktail.Adsorbed proteins are solubilized in sample buffer and separated on 12%SDS-PAGE minigels.

Example V

[0209] Northern Blot Analysis:

[0210] A 784 bp amplicon from the novel human kinase cDNA (SEQ ID NO:1base positions 128-911) was used as the hybridization probe againstCLONTECH Human Immune System Multiple Tissue Northern Blot II and HumanMultiple Tissue Northern Blot as sources for poly A+ RNA. Labeling ofthe probe was performed using the Ready-To-Go™ DNA Labeling Kit(Pharmacia) with 50 μCi [α-³²P] DATP. Hybridization was performedsubstantially as described by Clontech (Protocol # PT-1200-1) usingExpressHyb™ hybridization solution. FIG. 9 displays a Northern analysiswhich identifies a primary transcript pertaining to the novel humankinase, approximately 2.5 kilobases in length. Prominent transcripts areapparent, especially in immune tissues, heart, and skeletal muscle.

Example VI

[0211] High Throughput Screening for Compounds Which Modulate Activity:

[0212] High throughput screening for modulator compounds is performedusing MBP coated 96-well FlashPlates® (NEN™ Life Science Products).Kinase reaction buffer (3× kinase reaction buffer (KRB) contains: 60 mMHEPES (pH 7.5), 30 mM magnesium acetate, 0.15 mM ATP, 3 mM DTT, 0.03 mMsodium orthovanadate) 0.25 μCi [γ-³³P]-ATP at a concentration no greaterthan 1 μg/ml, (determined by titration of individual enzyme preparationsfor a concentration that allows kinetic determinations over a 1 hourtime course of the human kinase) are added to each well and incubated 1hour at 30° C. in the presence or absence of 10 μM test compound. Totalreaction volume is 100 μL. Following incubation, the reaction mixture isaspirated and the wells rinsed 2 times with 300 μL PBS. Incorporation ofradiolabeled phosphate is determined by scintillation counting, PackardInstrument Co. TopCount, 12-detector, 96-well microplate scintillationcounter and luminescence counter, model B991200. Compounds which inhibitkinase activity ≧50 percent at 10 μM are are indicated by a >50%reduction in scintillation counts. Specificity and selectivity studiesis determined by titration of inhibitory compounds to determine the IC₅₀(or other standard quantitation well known in the art for comparison)and by the substitution of other kinases in the assay. For example,determination of relative inhibitory activity of the kinase incomparison to recombinant SOK-1, expressed and isolated in a similarmanner, assayed under similar conditions, provides selectivity data.

Example VII

[0213] High Throughput Screening Protocol:

[0214] Test Compounds

[0215] Test compounds are prepared in advance from 2.5 mg/ml stocksolutions in DMSO by diluting 1:10 in distilled water and then 1:10again. Ten (1 0)μl of the 1:100 dilution solutions (25 μg/ml in 1% DMSO)are prepared in 96 well Microlite 1 plates (Dynatech) and plates arestored at −20° C. until the evening prior to the start of the assay.

[0216] Control Plates

[0217] A plate containing control solutions is included in each run ofthe screen for QA purposes. Such plates are prepared at the beginning ofthe HTS campaign and stored at −20° C. until required. Zero inhibition(MAX. signal) wells (columns 3, 6, 8 and 10) contain 10 μl of 1% (v/v)DMSO solution in MilliQ water. 100% inhibition (MIN signal) wells(columns 1, 4, 9 and 11) contain 10 μl of 220 nM ZM333141/1 in 1% DMSOsolution in MilliQ water. 50% inhibition (REF. signal) wells (columns 2,5, 7 and 12) contain a reference compound at a concentration known toprovide approximately 50% inhibition in 1% (v/v) DMSO solution in MilliQwater.

[0218] Assay Components

[0219] (1) recombinant kinase (expressed in E. coli or eukaryotic cellsas described herein) or a lysate of a prokaryotic or eukaryotic cellexpressing recombinant enzyme, or the natural enzyme partially purifiedfrom a human cell line.

[0220] (2) [γ-³³-P]-adenosine triphosphate

[0221] (3) myelin basic protein linked to the surface of PVT SPA beads(purchased from Amersham International) by an antibody-protein A orother appropriate method.

[0222] To Microlite I plates containing 10 μl of test compound, whichhave been left on the bench overnight to reach room temperature, 25 mlof GST-Rb/ATP/ATP³³ is added, immediately followed by 20 μl of Enzyme,using two Multidrops. The plates are stacked in 13 plate stacks (with anempty plate on top of each stack to minimise evaporation from the topplate) and left at room temperature for 105 minutes. 150 μl of “StopSolution” containing beads antibody and EDTA is added using a Multidrop.The plates are sealed with Topseal-S plate sealers and left on the benchovernight, surrounded by Scotlab perspex screens. The plates are thencentrifuged (Heraeus Megafuge 3.0R) at 2500 rpm, 1124× g., for 5 minutes(2 plates per trunnion) and counted on a Topcount (I4.34); (isotope:P³³;counting time: 20 seconds/well).

[0223] The data may be analysed using well-known software systems. Athreshold for inhibition is set, e.g., 60% inhibition of scintillationsignal. Compounds reaching the inhibition threshold are scored asactive.

Example VIII

[0224] PCR of SEQ ID NO:1 from Various Human cDNAS of HematopoieticOrigin:

[0225] SEQ ID NO:1 message is determined to be present in other cells ofhematopoietic origin by PCR with cDNA's isolated from differentcell-lines of hematopoietic origin.

[0226] Target product was amplified from 2-2.5 ng of reverse transcribedmRNAs in a 20 μL reaction using Advantage™ KlenTaq polymerase (Clontech# 8417-1, lot 7020348) according to the manufacturer's recommendations.Primer set 1 sequences are: 5′ CTGAAACACCGGAAGCTC 3′ (forward,corresponding to SEQ ID NO:1 nucleotides 1462-1479) and 5′ATGAGGGTATGCAGAGTGG 3′ (reverse, corresponding to SEQ ID NO:1nucleotides 1904-1922). Primer set 2 was the same set used to generateprobe for the cDNA library screening. Primer set 2 reactions includedDMSO at a final concentration of 5%. Cycling parameters were accordingto Clontech Marathon-Ready cDNA User Manual (PT1156-1) p. 19, program 1(briefly, touchdown PCR with 1.5-2 minute extensions). 10 μL of eachreaction was analyzed on a 1% agarose gel containing 0.5 μg/mL finalconcentration ethidium bromide in TAE buffer as in Sambrook et al.,Molecular Cloning Lab Manual, Second Edition, Cold Spring Harbor Press(1989), using 600 ng of 1 kb DNA ladder as markers (Life Technologies,cat # 15615-016).

[0227] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described methods and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 9 1 2322 DNA Homo Sapiens 1 gagcgccatg gctcactccc cggtgcagtcgggcctgccc ggcatgcaga acctaaaggc 60 agacccagaa gagcttttta caaaactagagaaaattggg aagggctcct ttggagaggt 120 gttcaaaggc attgacaatc ggactcagaaagtggttgcc ataaagatca ttgatctgga 180 agaagctgaa gatgagatag aggacattcaacaagaaatc acagtgctga gtcagtgtga 240 cagtccatat gtaaccaaat attatggatcctatctgaag gatacaaaat tatggataat 300 aatggaatat cttggtggag gctccgcactagatctatta gaacctggcc cattagatga 360 aacccagatc gctactatat taagagaaatactgaaagga ctcgattatc tccattcgga 420 gaagaaaatc cacagagaca ttaaagcggccaacgtcctg ctgtctgagc atggcgaggt 480 gaagctggcg gactttggcg tggctggccagctgacagac acccagatca aaaggaacac 540 cttcgtgggc accccattct ggatggcacccgaggtcatc aaacagtcgg cctatgactc 600 gaaggcagac atctggtccc tgggcataacagctattgaa cttgcaagag gggaaccacc 660 tcattccgag ctgcacccca tgaaagttttattcctcatt ccaaagaaca acccaccgac 720 gttggaagga aactacagta aacccctcaaggagtttgtg gaggcctgtt tgaataagga 780 gccgagcttt agacccactg ctaaggagttattgaagcac aagtttatac tacgcaatgc 840 aaagaaaact tcctacttga ccgagctcatcgacaggtac aagagatgga aggccgagca 900 gagccatgac gactcgagct ccgaggattccgacgcggaa acagatggcc aagcctcggg 960 gggcagtgat tctggggact ggatcttcacaatccgagaa aaagatccca agaatctcga 1020 gaatggagct cttcagccat cggacttggacagaaataag atgaaagaca tcccaaagag 1080 gcctttctct cagtgtttat ctacaattatttctcctctg tttgcagagt tgaaggagaa 1140 gagccaggcg tgcggaggga acttggggtccattgaagag ctgcgagggg ccatctacct 1200 agcggaggag gcgtgccctg gcatctccgacaccatggtg gcccagctcg tgcagcggct 1260 ccagagatac tctctaagtg gtggaggaacttcatcccac tgaaattcct ttggcatttg 1320 gggttttgtt tttccttttt tccttcttcatcctcctcct tttttaaaag tcaacgagag 1380 ccttcgctga ctccaccgaa gaggtgcgccactgggagcc accccagcgc caggcgcccg 1440 tccagggaca cacacagtct tcactgtgctgcagccagat gaagtctctc agatgggtgg 1500 ggagggtcag ctccttccag cgatcattttattttatttt attacttttg tttttaattt 1560 taaccatagt gcacatattc caggaaagtgtctttaaaaa caaaaacaaa ccctgaaatg 1620 tatatttggg attatgataa ggcaactaaagacatgaaac ctcaggtatc ctgctttaag 1680 ttgataactc cctctgggag ctggagaatcgctctggtgg atgggtgtac agatttgtat 1740 ataatgtcat ttttacggaa accctttcggcgtgcataag gaatcactgt gtacaaactg 1800 gccaagtgct tctgtagata acgtcagtggagtaaatatt cgacaggcca taacttgagt 1860 ctattgcctt gcctttatta catgtacattttgaattctg tgaccagtga tttgggtttt 1920 attttgtatt tgcagggttt gtcattaataattaatgccc ctctcttaca gaacactcct 1980 atttgtacct caacaaatgc aaattttccccgtttgccct acgccccttt tggtacacct 2040 agaggttgat ttcctttttc atcgatggtactatttctta gtgttttaaa ttggaacata 2100 tcttgcctca tgaagcttta aattataattttcagtttct ccccatgaag cgctctcgtc 2160 tgacatttgt ttggaatcgt gccactgctggtctgcgcca gatgtaccgt cctttccaat 2220 acgattttct gttgcacctt gtagtggattctgcatatca tctttcccac ctaaaaatgt 2280 ctgaatgctt acacaaataa attttataacacgcttaaaa aa 2322 2 1296 DNA Homo Sapiens 2 atggctcact ccccggtgcagtcgggcctg cccggcatgc agaacctaaa ggcagaccca 60 gaagagcttt ttacaaaactagagaaaatt gggaagggct cctttggaga ggtgttcaaa 120 ggcattgaca atcggactcagaaagtggtt gccataaaga tcattgatct ggaagaagct 180 gaagatgaga tagaggacattcaacaagaa atcacagtgc tgagtcagtg tgacagtcca 240 tatgtaacca aatattatggatcctatctg aaggatacaa aattatggat aataatggaa 300 tatcttggtg gaggctccgcactagatcta ttagaacctg gcccattaga tgaaacccag 360 atcgctacta tattaagagaaatactgaaa ggactcgatt atctccattc ggagaagaaa 420 atccacagag acattaaagcggccaacgtc ctgctgtctg agcatggcga ggtgaagctg 480 gcggactttg gcgtggctggccagctgaca gacacccaga tcaaaaggaa caccttcgtg 540 ggcaccccat tctggatggcacccgaggtc atcaaacagt cggcctatga ctcgaaggca 600 gacatctggt ccctgggcataacagctatt gaacttgcaa gaggggaacc acctcattcc 660 gagctgcacc ccatgaaagttttattcctc attccaaaga acaacccacc gacgttggaa 720 ggaaactaca gtaaacccctcaaggagttt gtggaggcct gtttgaataa ggagccgagc 780 tttagaccca ctgctaaggagttattgaag cacaagttta tactacgcaa tgcaaagaaa 840 acttcctact tgaccgagctcatcgacagg tacaagagat ggaaggccga gcagagccat 900 gacgactcga gctccgaggattccgacgcg gaaacagatg gccaagcctc ggggggcagt 960 gattctgggg actggatcttcacaatccga gaaaaagatc ccaagaatct cgagaatgga 1020 gctcttcagc catcggacttggacagaaat aagatgaaag acatcccaaa gaggcctttc 1080 tctcagtgtt tatctacaattatttctcct ctgtttgcag agttgaagga gaagagccag 1140 gcgtgcggag ggaacttggggtccattgaa gagctgcgag gggccatcta cctagcggag 1200 gaggcgtgcc ctggcatctccgacaccatg gtggcccagc tcgtgcagcg gctccagaga 1260 tactctctaa gtggtggaggaacttcatcc cactga 1296 3 431 PRT Homo Sapiens 3 Met Ala His Ser Pro ValGln Ser Gly Leu Pro Gly Met Gln Asn Leu 1 5 10 15 Lys Ala Asp Pro GluGlu Leu Phe Thr Lys Leu Glu Lys Ile Gly Lys 20 25 30 Gly Ser Phe Gly GluVal Phe Lys Gly Ile Asp Asn Arg Thr Gln Lys 35 40 45 Val Val Ala Ile LysIle Ile Asp Leu Glu Glu Ala Glu Asp Glu Ile 50 55 60 Glu Asp Ile Gln GlnGlu Ile Thr Val Leu Ser Gln Cys Asp Ser Pro 65 70 75 80 Tyr Val Thr LysTyr Tyr Gly Ser Tyr Leu Lys Asp Thr Lys Leu Trp 85 90 95 Ile Ile Met GluTyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Glu 100 105 110 Pro Gly ProLeu Asp Glu Thr Gln Ile Ala Thr Ile Leu Arg Glu Ile 115 120 125 Leu LysGly Leu Asp Tyr Leu His Ser Glu Lys Lys Ile His Arg Asp 130 135 140 IleLys Ala Ala Asn Val Leu Leu Ser Glu His Gly Glu Val Lys Leu 145 150 155160 Ala Asp Phe Gly Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Lys Arg 165170 175 Asn Thr Phe Val Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Lys180 185 190 Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Trp Ser Leu Gly IleThr 195 200 205 Ala Ile Glu Leu Ala Arg Gly Glu Pro Pro His Ser Glu LeuHis Pro 210 215 220 Met Lys Val Leu Phe Leu Ile Pro Lys Asn Asn Pro ProThr Leu Glu 225 230 235 240 Gly Asn Tyr Ser Lys Pro Leu Lys Glu Phe ValGlu Ala Cys Leu Asn 245 250 255 Lys Glu Pro Ser Phe Arg Pro Thr Ala LysGlu Leu Leu Lys His Lys 260 265 270 Phe Ile Leu Arg Asn Ala Lys Lys ThrSer Tyr Leu Thr Glu Leu Ile 275 280 285 Asp Arg Tyr Lys Arg Trp Lys AlaGlu Gln Ser His Asp Asp Ser Ser 290 295 300 Ser Glu Asp Ser Asp Ala GluThr Asp Gly Gln Ala Ser Gly Gly Ser 305 310 315 320 Asp Ser Gly Asp TrpIle Phe Thr Ile Arg Glu Lys Asp Pro Lys Asn 325 330 335 Leu Glu Asn GlyAla Leu Gln Pro Ser Asp Leu Asp Arg Asn Lys Met 340 345 350 Lys Asp IlePro Lys Arg Pro Phe Ser Gln Cys Leu Ser Thr Ile Ile 355 360 365 Ser ProLeu Phe Ala Glu Leu Lys Glu Lys Ser Gln Ala Cys Gly Gly 370 375 380 AsnLeu Gly Ser Ile Glu Glu Leu Arg Gly Ala Ile Tyr Leu Ala Glu 385 390 395400 Glu Ala Cys Pro Gly Ile Ser Asp Thr Met Val Ala Gln Leu Val Gln 405410 415 Arg Leu Gln Arg Tyr Ser Leu Ser Gly Gly Gly Thr Ser Ser His 420425 430 4 426 PRT Homo Sapiens 4 Met Ala His Leu Arg Gly Phe Ala Asn GlnHis Ser Arg Val Asp Pro 1 5 10 15 Glu Glu Leu Phe Thr Lys Leu Asp ArgIle Gly Lys Gly Ser Phe Gly 20 25 30 Glu Val Tyr Lys Gly Ile Asp Asn HisThr Lys Glu Val Val Ala Ile 35 40 45 Lys Ile Ile Asp Leu Glu Glu Ala GluAsp Glu Ile Glu Asp Ile Gln 50 55 60 Gln Glu Ile Thr Val Leu Ser Gln CysAsp Ser Pro Tyr Ile Thr Arg 65 70 75 80 Tyr Phe Gly Ser Tyr Leu Lys SerThr Lys Leu Trp Ile Ile Met Glu 85 90 95 Tyr Leu Gly Gly Gly Ser Ala LeuAsp Leu Leu Lys Pro Gly Pro Leu 100 105 110 Glu Glu Thr Tyr Ile Ala ThrIle Leu Arg Glu Ile Leu Lys Gly Leu 115 120 125 Asp Tyr Leu His Ser GluArg Lys Ile His Arg Asp Ile Lys Ala Ala 130 135 140 Asn Val Leu Leu SerGlu Gln Gly Asp Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Val Ala GlyGln Leu Thr Asp Thr Gln Ile Lys Arg Asn Thr Phe Val 165 170 175 Gly ThrPro Phe Trp Met Ala Pro Glu Val Ile Lys Gln Ser Ala Tyr 180 185 190 AspPhe Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile Glu Leu 195 200 205Ala Lys Gly Glu Pro Pro Asn Ser Asp Leu His Pro Met Arg Val Leu 210 215220 Phe Leu Ile Pro Lys Asn Ser Pro Pro Thr Leu Glu Gly Gln His Ser 225230 235 240 Lys Pro Phe Lys Glu Phe Val Glu Ala Cys Leu Asn Lys Asp ProArg 245 250 255 Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys His Lys Phe IleThr Arg 260 265 270 Tyr Thr Lys Lys Thr Ser Phe Leu Thr Glu Leu Ile AspArg Tyr Lys 275 280 285 Arg Trp Lys Ser Glu Gly His Gly Glu Glu Ser SerSer Glu Asp Ser 290 295 300 Asp Ile Asp Gly Glu Ala Glu Asp Gly Glu GlnGly Pro Ile Trp Thr 305 310 315 320 Phe Pro Pro Thr Ile Arg Pro Ser ProHis Ser Lys Leu His Lys Gly 325 330 335 Thr Ala Leu His Ser Ser Gln LysPro Ala Asp Ala Val Lys Arg Gln 340 345 350 Pro Arg Ser Gln Cys Leu SerThr Leu Val Arg Pro Val Phe Gly Glu 355 360 365 Leu Lys Glu Lys His LysGln Ser Gly Gly Ser Val Gly Ala Leu Glu 370 375 380 Glu Leu Glu Asn AlaPhe Ser Leu Ala Glu Glu Ser Cys Pro Gly Ile 385 390 395 400 Ser Asp LysLeu Met Val His Leu Val Glu Arg Val Gln Arg Phe Ser 405 410 415 His AsnArg Asn His Leu Thr Ser Thr Arg 420 425 5 2322 DNA Artificial SequenceAntisense 5 ttttttaagc gtgttataaa atttatttgt gtaagcattc agacatttttaggtgggaaa 60 gatgatatgc agaatccact acaaggtgca acagaaaatc gtattggaaaggacggtaca 120 tctggcgcag accagcagtg gcacgattcc aaacaaatgt cagacgagagcgcttcatgg 180 ggagaaactg aaaattataa tttaaagctt catgaggcaa gatatgttccaatttaaaac 240 actaagaaat agtaccatcg atgaaaaagg aaatcaacct ctaggtgtaccaaaaggggc 300 gtagggcaaa cggggaaaat ttgcatttgt tgaggtacaa ataggagtgttctgtaagag 360 aggggcatta attattaatg acaaaccctg caaatacaaa ataaaacccaaatcactggt 420 cacagaattc aaaatgtaca tgtaataaag gcaaggcaat agactcaagttatggcctgt 480 cgaatattta ctccactgac gttatctaca gaagcacttg gccagtttgtacacagtgat 540 tccttatgca cgccgaaagg gtttccgtaa aaatgacatt atatacaaatctgtacaccc 600 atccaccaga gcgattctcc agctcccaga gggagttatc aacttaaagcaggatacctg 660 aggtttcatg tctttagttg ccttatcata atcccaaata tacatttcagggtttgtttt 720 tgtttttaaa gacactttcc tggaatatgt gcactatggt taaaattaaaaacaaaagta 780 ataaaataaa ataaaatgat cgctggaagg agctgaccct ccccacccatctgagagact 840 tcatctggct gcagcacagt gaagactgtg tgtgtccctg gacgggcgcctggcgctggg 900 gtggctccca gtggcgcacc tcttcggtgg agtcagcgaa ggctctcgttgacttttaaa 960 aaaggaggag gatgaagaag gaaaaaagga aaaacaaaac cccaaatgccaaaggaattt 1020 cagtgggatg aagttcctcc accacttaga gagtatctct ggagccgctgcacgagctgg 1080 gccaccatgg tgtcggagat gccagggcac gcctcctccg ctaggtagatggcccctcgc 1140 agctcttcaa tggaccccaa gttccctccg cacgcctggc tcttctccttcaactctgca 1200 aacagaggag aaataattgt agataaacac tgagagaaag gcctctttgggatgtctttc 1260 atcttatttc tgtccaagtc cgatggctga agagctccat tctcgagattcttgggatct 1320 ttttctcgga ttgtgaagat ccagtcccca gaatcactgc cccccgaggcttggccatct 1380 gtttccgcgt cggaatcctc ggagctcgag tcgtcatggc tctgctcggccttccatctc 1440 ttgtacctgt cgatgagctc ggtcaagtag gaagttttct ttgcattgcgtagtataaac 1500 ttgtgcttca ataactcctt agcagtgggt ctaaagctcg gctccttattcaaacaggcc 1560 tccacaaact ccttgagggg tttactgtag tttccttcca acgtcggtgggttgttcttt 1620 ggaatgagga ataaaacttt catggggtgc agctcggaat gaggtggttcccctcttgca 1680 agttcaatag ctgttatgcc cagggaccag atgtctgcct tcgagtcataggccgactgt 1740 ttgatgacct cgggtgccat ccagaatggg gtgcccacga aggtgttccttttgatctgg 1800 gtgtctgtca gctggccagc cacgccaaag tccgccagct tcacctcgccatgctcagac 1860 agcaggacgt tggccgcttt aatgtctctg tggattttct tctccgaatggagataatcg 1920 agtcctttca gtatttctct taatatagta gcgatctggg tttcatctaatgggccaggt 1980 tctaatagat ctagtgcgga gcctccacca agatattcca ttattatccataattttgta 2040 tccttcagat aggatccata atatttggtt acatatggac tgtcacactgactcagcact 2100 gtgatttctt gttgaatgtc ctctatctca tcttcagctt cttccagatcaatgatcttt 2160 atggcaacca ctttctgagt ccgattgtca atgcctttga acacctctccaaaggagccc 2220 ttcccaattt tctctagttt tgtaaaaagc tcttctgggt ctgcctttaggttctgcatg 2280 ccgggcaggc ccgactgcac cggggagtga gccatggcgc tc 2322 6 46DNA Artificial Sequence Primer 6 ggactcagaa agtggttgcc attcgaataattgatctgga agaagc 46 7 46 DNA Artificial Sequence Primer 7 gcttcttccagatcaattat tcgaatggca accactttct gagtcc 46 8 19 DNA Artificial SequencePrimer 8 gagcgccatg gctcactcc 19 9 20 DNA Artificial Sequence Primer 9ggagtcagcg aaggctctcg 20

1. A purified polynucleotide comprising a nucleic acid sequence whichencodes a polypeptide comprising a sequence selected from the groupconsisting of a sequence at least 90% similar to SEQ ID NO: 3 and asequence at least 90% identical to residues 24-295 of SEQ ID NO:
 3. 2.The polynucleotide of claim 1, comprising the sequence disclosed in SEQID NO:
 2. 3. An expression vector comprising a polynucleotide encodes apolypeptide at least 90% similar to SEQ ID NO: 3 according to claim 1.4. A host cell transformed with the expression vector of claim
 3. 5. Amethod for producing cells which express a polypeptide at least 90%similar to SEQ ID NO: 3, said method comprising culturing a host cellaccording to claim 4 in culture medium under conditions suitable forgrowth of said host cell.
 6. A method for producing a polypeptide atleast 90% similar to SEQ ID NO: 3, said method comprising the steps of:a) culturing said host cell according to claim 4 in culture medium underconditions suitable for the expression of said polypeptide; and, b)recovering said polypeptide from cultured host cells or said culturemedium.
 7. An expression vector comprising a polynucleotide sequencewhich encodes a polypeptide at least 90% identical to residues 24-295 ofSEQ ID NO: 3 according to claim
 1. 8. A host cell transformed with theexpression vector of claim
 7. 9. A method for producing cells whichexpress a polypeptide at least 90% identical to residues 24-295 of SEQID NO: 3, said method comprising culturing a host cell according toclaim 8 in culture medium under conditions suitable for growth of saidhost cell.
 10. A method for producing a polypeptide at least 90%identical to residues 24-295 of SEQ ID NO: 3, said method comprising thesteps of: a) culturing said host cell according to claim 8 in culturemedium under conditions suitable for the expression of said polypeptide;and, b) recovering said polypeptide from cultured host cells or saidculture medium.
 11. A purified polynucleotide comprising a nucleic acidsequence which encodes a polypeptide comprising a sequence at least 90%similar to SEQ ID NO:
 3. 12. A purified polynucleotide comprising anucleic acid sequence which encodes a polypeptide comprising a sequenceat least 90% identical residues 24-295 of SEQ ID NO: 3.